WO2010087306A1 - Anti-neurodegenerative disease agent - Google Patents

Anti-neurodegenerative disease agent Download PDF

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Publication number
WO2010087306A1
WO2010087306A1 PCT/JP2010/050903 JP2010050903W WO2010087306A1 WO 2010087306 A1 WO2010087306 A1 WO 2010087306A1 JP 2010050903 W JP2010050903 W JP 2010050903W WO 2010087306 A1 WO2010087306 A1 WO 2010087306A1
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Prior art keywords
inhibitor
agent
test
compound represented
neurodegenerative disease
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PCT/JP2010/050903
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French (fr)
Japanese (ja)
Inventor
人水 太田
研志 穐田
恒孝 太田
河田　敏雄
福田　恵温
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株式会社林原生物化学研究所
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Application filed by 株式会社林原生物化学研究所 filed Critical 株式会社林原生物化学研究所
Priority to EP10735774.1A priority Critical patent/EP2397139B1/en
Priority to JP2010548497A priority patent/JP5591720B2/en
Priority to US13/146,840 priority patent/US20120035187A1/en
Publication of WO2010087306A1 publication Critical patent/WO2010087306A1/en
Priority to US14/097,566 priority patent/US20140094490A1/en

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Definitions

  • the present invention relates to an anti-neurodegenerative disease agent comprising a compound represented by the general formula 1 as an active ingredient.
  • R 1 to R 3 in General Formula 1 each independently represent a hydrogen atom or an appropriate substituent
  • Z 1 represents a heterocyclic ring
  • Z 2 represents a heterocyclic ring or an aromatic ring that is the same as or different from Z 1.
  • These heterocyclic rings and aromatic rings may have a substituent.
  • o represents an integer that is either 0, 1, or 2
  • p represents an integer that is either 0 or 1
  • p is 1 when o is 0 or 2
  • o is 1
  • P is 0.
  • X l - represents a suitable counter anion
  • q is an integer of either 1 or 2.
  • Neurodegenerative diseases are pathological conditions caused by systematic neuronal degeneration and disruption of the neural network based on loss, and many intractable diseases such as Alzheimer's disease, Parkinson's disease, Parkinson's syndrome, cerebrovascular dementia, frontal side Known as lobar dementia, amyotrophic lateral sclerosis, progressive supranuclear palsy, Huntington's disease, spinocerebellar degeneration, and the like.
  • NGF nerve growth factor
  • Clinical symptoms of neurodegenerative diseases vary depending on each disease, but they vary from minor to severe. Typical examples include tremor, rigidity, agitation, peristalsis, and movement. Slowness, postural reflex disorder, autonomic disorder, lunging phenomenon, gait disorder, depression, memory disorder, muscle atrophy, muscle weakness, upper limb dysfunction, articulation disorder, dysphagia, respiratory disorder, numbness or paralysis, etc. Both are major obstacles in daily life.
  • Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are serious diseases that cause degeneration of nerve cells, and various compounds are used as active ingredients to improve these diseases and their associated pathological conditions and neurological dysfunction.
  • International Publication No. WO97 / 030703, JP-A-11-228417, JP-A-2006-143708, and JP-A-2006-321737 Although an accelerator or the like has also been proposed (see, for example, Japanese Patent Application Laid-Open No. 2002-234841), an effective disease treatment method has not yet been found.
  • commercially available therapeutic agents for neurodegenerative diseases may have problems in terms of side effects and the like for long-term continuous use.
  • systemic administration such as subcutaneous or intravascular, which has less physical and mental burden for patients, acts on nerve cells of the central nervous system, activates nerve cells, suppresses neurite atrophy, or Development of a novel anti-neurodegenerative disease agent capable of suppressing neurite outgrowth to suppress neurodegeneration and treating the pathological conditions and clinical symptoms associated therewith is eagerly desired.
  • An object of the present invention is to provide a novel anti-neurodegenerative disease agent.
  • the present inventors have conducted extensive research and search.
  • the compound represented by the following general formula 1 has an excellent nerve cell activation action and a neurite extension promoting action. I found out.
  • these compounds have an inhibitory effect on neuronal cell death caused by cytotoxic factors, and even when administered systemically, they activate neurons in the central nervous system to suppress neurodegeneration, as well as symptoms and pathologies caused by neurodegeneration.
  • the present invention was completed by finding that the onset was delayed or improved. That is, the present invention mainly comprises an anti-neurodegenerative disease agent containing a compound represented by the following general formula 1 as an active ingredient.
  • R 1 to R 3 in General Formula 1 each independently represent a hydrogen atom or an appropriate substituent
  • Z 1 represents a heterocyclic ring
  • Z 2 represents a heterocyclic ring or an aromatic ring that is the same as or different from Z 1.
  • These heterocyclic rings and aromatic rings may have a substituent.
  • o represents an integer that is either 0, 1, or 2
  • p represents an integer that is either 0 or 1
  • p is 1 when o is 0 or 2
  • o is 1
  • P is 0.
  • X l - represents a suitable counter anion
  • q is an integer of either 1 or 2.
  • the anti-neurodegenerative disease agent of the present invention promotes nerve cell proliferation and neurite outgrowth by parenteral administration, and at the same time removes cells from cytotoxic factors such as nutrient and oxygen starvation and amyloid ⁇ peptide.
  • cytotoxic factors such as nutrient and oxygen starvation and amyloid ⁇ peptide.
  • security of the compound represented by General formula 1 which is an active ingredient is very high.
  • the neurite means an axon and dendrite extending from a neuronal cell body.
  • the neurite outgrowth promoting action refers to the action of activating nerve cells to extend axons and / or dendrites, the action of suppressing neurite atrophy and decrease, and the action of promoting synapse formation between nerve cells. And the action of suppressing the decrease in synapses.
  • Neurodegeneration as used in the present invention refers to a decrease in function, death, or decrease (dropout) of nerve cells, particularly neurons in the central nervous system. Atrophy and decrease of neurites, decrease of synapses, decrease of function of glial cells , Including death and reduction, death and degeneration of retinal cells.
  • the anti-neurodegenerative disease agent of the present invention contains a compound represented by the following general formula 1 as an active ingredient.
  • R 1 to R 3 in General Formula 1 each independently represent a hydrogen atom or an appropriate substituent
  • Z 1 represents a heterocyclic ring
  • Z 2 represents a heterocyclic ring or an aromatic ring that is the same as or different from Z 1.
  • These heterocyclic rings and aromatic rings may have a substituent.
  • o represents an integer that is either 0, 1, or 2
  • p represents an integer that is either 0 or 1
  • p is 1 when o is 0 or 2
  • o is 1
  • P is 0.
  • X l - represents a suitable counter anion
  • q is an integer of either 1 or 2.
  • X l in the general formula 1 - represents an appropriate counter anion, usually, for example, fluorine ion, chlorine ion, bromine ion, iodine ion, perchlorate ion, periodic acid ion, hexafluorophosphate ion, Rokudoruka Inorganic acid anions such as antimonate ions, hexafluorostannate ions, phosphate ions, borofluoride ions, tetrafluoroborate ions, thiocyanate ions, benzenesulfonate ions, naphthalenesulfonate ions, naphthalene disulfonate ions Organic acid anions such as p-toluenesulfonate ion, alkylsulfonate ion, benzenecarboxylate ion, alkylcarboxylate ion, trihaloalkylcarboxylate ion,
  • examples of the compound represented by the general formula 1 include dye compounds such as a pentamethine cyanine dye represented by any one of the general formulas 2 to 4 and a dimethine styryl dye represented by the general formula 5 ( Hereinafter, it may be simply referred to as “compound”).
  • R 4 to R 6 represent the same or different aliphatic hydrocarbon groups.
  • X 2 ⁇ represents an appropriate counter anion
  • m represents an integer that is either 1 or 2 that has a charge balanced with the charge of the cation moiety.
  • R 7 to R 9 represent the same or different aliphatic hydrocarbon groups.
  • X 3 - represents an appropriate counter anion
  • m represents an integer of either charge become 1 or 2 to balance the charge of the cation.
  • R 10 to R 12 represent the same or different aliphatic hydrocarbon groups.
  • X 4 ⁇ represents an appropriate counter anion
  • m represents an integer that is either 1 or 2 that has a charge balanced with the charge of the cation moiety.
  • Z 3 represents a heteroaromatic ring, and the heteroaromatic ring may have a substituent.
  • Z 4 represents an aromatic ring or a heteroaromatic ring, and the heteroaromatic ring and the aromatic ring may have a substituent.
  • R 13 represents an aliphatic hydrocarbon group, and the aliphatic hydrocarbon group may have a substituent.
  • the R 14 is hydrogen atom or an appropriate substituent and, X 5 - represents a suitable counter anion.
  • the aliphatic hydrocarbon group represented by R 4 to R 13 in the general formulas 2 to 5 those having 1 to 12 carbon atoms are usually selected, those having 2 to 10 are preferable, and those having 2 to 9 are preferable. Those are more preferred. Among them, the aliphatic hydrocarbon group of R 4 to R 6 of the compound represented by the general formula 2 has 2 to 12 carbon atoms, or the compound of the compound represented by the general formula 3 of R 7 to R 9 is aliphatic. A compound having 4 to 10 carbon atoms in the hydrocarbon group is particularly desirable because it has a strong inhibitory effect on neurodegeneration.
  • Examples of the individual aliphatic hydrocarbon group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, and a tert-pentyl group.
  • suitable counter anions represented by X 2 ⁇ to X 5 ⁇ in the general formulas 2 to 5 are usually, for example, fluorine ion, chlorine ion, bromine ion, iodine ion, perchlorate ion, periodate ion , Inorganic acid anions such as hexafluorophosphate ion, hexafluoroantimonate ion, hexafluorostannate ion, phosphate ion, borofluoride ion, tetrafluoroborate ion, thiocyanate ion, benzenesulfonate ion , Naphthalene sulfonate ion, naphthalene disulfonate ion, p-toluene sulfonate ion, alkyl sulfonate ion, benzene carboxylate ion, alkyl carboxylate ion,
  • the compound represented by the general formula 2 examples include a compound represented by the chemical formula 1 (hereinafter sometimes referred to as “NK-26”) and a compound represented by the chemical formula 2 (hereinafter, referred to as “NK-26”). Or a compound having 3 carbon atoms in the side chain alkyl group (R 4 to R 6 ) of the general formula 2 (hereinafter sometimes referred to as “NK-234”). It can be illustrated.
  • the structure of the compound corresponding to the NK number in this specification is, for example, “Photosensitive Dye Table”, published by Photosensitive Dye Research Institute (1969), or “Chemical Abstract Index Guide” (N- Z) ”, pages 1531G to 1536G (1994).
  • NK-150 a compound represented by the chemical formula 3
  • NK-150 a compound represented by the chemical formula 4
  • NK-19 may be mentioned.
  • the counter anion of the NK-19 (I -) of Cl - in the compound represented by Chemical Formula 5 for changing (. Which hereinafter may be referred to as "NK-53"), similar to the NK-19 advantageously Available.
  • NK-100 a compound represented by the chemical formula 6
  • the compound represented by the general formula 5 may be a compound represented by any one of the chemical formulas 7 to 9 (hereinafter referred to as “NK-528”, “NK-557”, and “NK-1516”, respectively). ).
  • the compounds represented by any one of Chemical Formulas 1 to 9 protect nerve cells from cytotoxic factors such as starvation, radicals, and amyloid ⁇ peptide, in addition to the nerve cell activation action and neurite outgrowth promoting action. Therefore, it is more desirable as an active ingredient of the anti-neurodegenerative disease agent of the present invention because of its action to suppress cell death and neurite atrophy.
  • NK-26 chemical formula 1
  • NK-4 compound represented by chemical formula 2)
  • NK-234 compound with 3 carbon atoms in the side chain alkyl group of general formula 2
  • NK-150 chemical formula 3
  • the compound represented is particularly desirable.
  • NK-4 and NK-234 are desirable, and NK-4 is particularly desirable, taking into account the strength of acetylcholinesterase (AchE) activity inhibitory activity, migration into the brain, ease of formulation, and the like.
  • AchE acetylcholinesterase
  • the compound represented by the general formula 1 used as an active ingredient of the anti-neurodegenerative disease agent of the present invention is not limited in its origin or production method.
  • the anti-neurodegenerative disease agent of the present invention is a compound represented by the general formula 1, preferably a pentamethine cyanine dye represented by any one of the general formulas 2 to 4 and / or a dimethine represented by the general formula 5. It contains one or more styryl dyes.
  • the anti-neurodegenerative disease agent of the present invention if necessary, in addition to the compound represented by the general formula 1 which is an active ingredient, is pharmaceutically acceptable in the food field, cosmetic field, pharmaceutical field, quasi drug It is provided in the form of a preparation containing one or more components used in the product field.
  • Examples of pharmaceutically acceptable ingredients include additives, excipients, disintegrants, lubricants, stabilizers, surfactants, preservatives (antibacterial agents), fragrances, thickeners, and antioxidants.
  • blend combining suitably 1 type (s) or 2 or more types of these components, and to manufacture by a conventional method according to the target dosage form.
  • the anti-neurodegenerative disease agent of the present invention is also advantageously used in combination with a neurite outgrowth promoting agent other than the compound represented by the general formula 1, or a therapeutic agent for a neurodegenerative disease, a pathological condition or a neurological disorder resulting therefrom. Can be implemented.
  • cerebrovascular disorders eg, stroke, cerebral infarction (eg, cerebral thrombus, cerebral embolism, etc.), transient ischemic attack, reperfusion injury, cerebral hemorrhage (eg, hypertensive intracerebral hemorrhage, arachnoid membrane) Etc.), brain tumors (eg, astrocytoma, brain abscess, etc.), blood volume reducing shock, traumatic shock, head injury and / or cerebrospinal trauma (eg, brain contusion, penetration, shear) ⁇
  • Therapeutic agents for neurological dysfunction associated with compression / laceration, labor trauma, infant whiplash, etc. neurodegenerative diseases (eg Parkinson's disease, Parkinson's syndrome, striatal nigra degeneration, Huntington's disease, chorea) Ataxia, progressive supranuclear palsy, diffuse Lewy body disease, basal ganglia degeneration, Alzheimer's disease, senile dementia, Pick's disease, frontotemporal
  • therapeutic agents for neurodegenerative diseases used in combination with the anti-neurodegenerative disease agent of the present invention and the pathological conditions and neurological dysfunctions resulting therefrom, preferably, for example, cerebrovascular disorders (eg, stroke, cerebral infarction (eg, cerebral thrombus) , Cerebral embolism etc.), transient cerebral ischemic attack, cerebral hemorrhage (eg hypertensive intracerebral hemorrhage, subarachnoid hemorrhage etc.), therapeutic agent for brain tumor, cerebrospinal trauma (eg cerebral contusion etc.
  • cerebrovascular disorders eg, stroke, cerebral infarction (eg, cerebral thrombus) , Cerebral embolism etc.
  • transient cerebral ischemic attack eg, cerebral hemorrhage (eg hypertensive intracerebral hemorrhage, subarachnoid hemorrhage etc.)
  • therapeutic agent for brain tumor eg cerebral contusion etc.
  • Drugs for neurological dysfunction associated with neurodegenerative diseases (eg Parkinson's disease, Parkinson's syndrome, Huntington's disease, Alzheimer's disease, senile dementia, spinocerebellar degeneration), motor neuropathy (eg muscle atrophy)
  • Treatment for demyelinating diseases eg, multiple sclerosis
  • cerebrospinal disease associated with infection eg, meningitis, influenza encephalopathy, Creutzfeldt-Jako) Disease, dementia due to AIDS encephalopathy, etc.
  • psychiatric disorders eg, neurosis, psychosomatic disorders, anxiety, schizophrenia, manic depression, etc.
  • epilepsy dystonia, diabetes , Diabetic complications and / or hyperlipidemia
  • dopamine receptor agonist dopamine release promoter
  • dopamine uptake inhibitor dopamine agonist
  • monoamine oxidase (MAO-B) inhibitor catechol-O-methyltrans
  • Therapeutics for diseases eg Parkinson's disease, Parkinson's syndrome, Huntington's disease, Alzheimer's disease, senile dementia, etc.
  • amyotrophic lateral sclerosis multiple sclerosis
  • mental illness eg Neurosis, psychosomatic disorders, anxiety, schizophrenia, manic depression, etc.
  • epilepsy and / or dystonia diabetes
  • Drugs for complications and / or drugs for hyperlipidemia dopamine receptor agonists, dopamine release promoters, dopamine uptake inhibitors, dopamine agonists, central anticholinergics, aromatic L-amino acid decarboxylase Inhibitor (DCI), monoamine oxidase (MAO-B) inhibitor, catechol-O-methyltransferase (COMT) inhibitor, norepinephrine (noradrenaline) supplement, acetylcholinesterase inhibitor, NMDA (N-methyl-D-asparagine) Acid) receptor antagonist, ⁇ -secretase inhibitor
  • the anti-neurodegenerative disease agent of the present invention is usually provided in the form of a parenteral injection preparation or the like.
  • the compound represented by the general formula 1, which is an active ingredient, is a process from the raw material stage to the completion of the product in consideration of the composition of the parenteral preparation such as an injectable preparation and the purpose of use. What is necessary is just to mix
  • the methods include, for example, mixing, kneading, dissolving, melting, dispersing, suspending, emulsifying, reverse micellization, infiltration, crystallization, spraying, application, adhesion, spraying, coating (coating), pouring, dipping, solidifying, One or more methods such as loading are appropriately selected.
  • parenteral preparations such as injection preparations
  • parenteral preparations such as injection preparations
  • it is usually dissolved in an aqueous medium that does not contain pyrogen, depending on the target disease or symptom, and then intradermally, subcutaneously, intramuscularly, intracorporeally (intrathoracic) , Intraperitoneal, etc.), intravascular or intracerebral (including spinal cord)
  • the preparation may be a dry preparation or a liquid preparation.
  • a dry preparation it may be used by dissolving it in an aqueous medium such as purified water for injection, physiological saline, and glucose solution at the time of use.
  • liquid preparation In the case of a liquid preparation, it may be administered as it is, or it may be added to an infusion solution, a perfusion solution, a peritoneal dialysis solution, or the like.
  • a liquid preparation when there is a problem with solubility in a solvent or solubility in an aqueous medium, or when preparing a sustained-release preparation, it is effective to use an amphiphilic solvent, an oily base material, an emulsifier, etc. Increasing the solubility of the components in the solvent is optional. It is also optional to encapsulate and administer in liposomes.
  • the aqueous medium referred to in the present invention has water as an essential element, and if necessary, for example, alcohols such as ethanol, propanol and isopropanol, ketones such as acetone, ethers such as diethyl ether, dimethyl It means a general aqueous medium comprising one or more hydrophilic organic solvents including sulfur-containing compounds such as sulfoxide (hereinafter sometimes abbreviated as “DMSO”).
  • DMSO sulfur-containing compounds
  • DMSO sulfur-containing compounds
  • aqueous solvent in the liquid preparation As the aqueous solvent in the liquid preparation according to the present invention, purified water for injection, physiological saline, Ringer's solution or the like is used alone, or purified water for injection and, for example, ethanol, propanol, isopropanol, diethyl ether, DMSO, etc. It is desirable to use a mixture with a physiologically acceptable hydrophilic organic solvent. It is also optional to add a pH adjuster such as lactic acid, hydrochloric acid, sodium hydroxide, potassium hydroxide, sodium bicarbonate or phosphate buffer to adjust the pH to the highest solubility of the compound to be formulated. .
  • a pH adjuster such as lactic acid, hydrochloric acid, sodium hydroxide, potassium hydroxide, sodium bicarbonate or phosphate buffer to adjust the pH to the highest solubility of the compound to be formulated.
  • Such a liquid agent depending on the compound represented by the general formula 1 used, it may become unstable due to dissolved oxygen or the like. In that case, for example, if the dissolved oxygen concentration of the compound solution is reduced, Good.
  • Such a liquid composition is usually prepared by a method through a step of dissolving the compound in an aqueous medium and a step of lowering the oxygen concentration in the atmospheric environment at normal temperature and pressure using the aqueous medium. be able to.
  • a predetermined amount of a compound is added to an appropriate amount of an aqueous medium, dissolved as necessary with heating and stirring, and then, if necessary, An aqueous medium may be added until the concentration reaches a predetermined level.
  • the compound solution represented by the general formula 1 is prepared under reduced pressure and stored or dissolved in the compound solution. It is preferable to replace oxygen with another gas or to bring the compound solution into contact with an oxygen scavenger.
  • a relatively inert gas such as nitrogen or a rare gas such as neon, argon, krypton, or xenon is used. Just bubbling.
  • the liquid composition is prepared by, for example, adding L-ascorbic acid, L-ascorbic acid stearate, sodium sulfite, sodium hydrogen sulfite, alphathioglycerin, sodium edetate, cysteine hydrochloride Citric acid, soybean lecithin, sodium thioglycolate, sodium thiomalate, sodium pyrosulfite, butylhydroxyanisole and the like may be added in appropriate amounts. These methods may be applied to the compound solution or to an aqueous medium before the compound is dissolved.
  • the concentration of oxygen dissolved in the aqueous medium is usually 0.4 ppm or less, preferably 0.1 ppm or less. It also has singlet oxygen scavenging activity such as tocopherol, carotene, histidine, tryptophan, tyrosine, methionine, cysteine, dopa, rutin, rutin derivatives, thiotaurine, hypotaurine, bilirubin, cholesterol, quinoline, quercetin, catechin, anthocyanin, thiamine, etc.
  • singlet oxygen scavenging activity such as tocopherol, carotene, histidine, tryptophan, tyrosine, methionine, cysteine, dopa, rutin, rutin derivatives, thiotaurine, hypotaurine, bilirubin, cholesterol, quinoline, quercetin, catechin, anthocyanin, thiamine, etc.
  • surfactants such as thickeners such as alkyl cellulose and carboxyl polymer, Triton X, polysorbate, deoxycholic acid or salts thereof, and cholic acid or salts thereof It is also possible to advantageously carry out the preparation by adding an appropriate amount for stabilization.
  • the thus obtained solution of the compound represented by the general formula 1 may be stored in a state where it is sealed in an appropriate container that can block oxygen.
  • the material of the container is not particularly limited as long as it can hold the liquid composition in principle and can substantially block oxygen, but it is light-shielded like a brown bottle or brown ampoule. Sex containers are desirable. Although it depends on the application, sterilization such as filtration sterilization is usually performed before dispensing the liquid composition into containers such as glass ampoules and vials, or after dispensing and sealing the container, high-pressure sterilization or filtration sterilization is performed. Sterilize by etc.
  • the anti-neurodegenerative disease agent of the present invention can be used in the form of a haptic agent, a transpulmonary sucking and spraying agent, etc. Can also be used. It is also optional to treat animals other than humans, including pets that have developed neurodegenerative diseases, and to use as preventives or therapeutic agents for pathological conditions and neurological dysfunction associated with neurodegenerative diseases.
  • the anti-neurodegenerative disease agent of the present invention thus produced is a safe preparation without serious side effects even when used for a long time.
  • the anti-neurodegenerative disease agent of the present invention is divided into once or multiple times per day at intervals of daily to 1 day or more depending on the target neurodegenerative disease, its pathology and symptoms. May be administered in a predetermined amount.
  • the daily dose is not particularly limited as long as the desired action and effect can be obtained, and is generally expressed by the general formula 1 in the case of intravenous administration (including infusion) and subcutaneous to intraperitoneal administration.
  • 0.01 mg / kg ⁇ body weight / day or more is desirable, 0.1 to 20 mg / kg ⁇ body weight / day is more desirable, and 0.5 to 5 mg / kg ⁇ body weight / day is particularly desirable.
  • the enhancement of the effect corresponding to the dose is not observed.
  • the administration period of the anti-neurodegenerative disease agent of the present invention may be prepared according to the target disease, condition or symptom, and may be administered until the symptom is improved or disappeared in the case of an acute disease. In the case of chronic diseases such as dementia, it is desirable to continue administration even if improvement or disappearance of symptoms is observed.
  • the anti-neurodegenerative disease agent of the present invention protects the brain and nerve cells from injury factors and suppresses degeneration, activates nerve cells, promotes neurite extension and suppresses atrophy, prolongs survival and degeneration of nerve cells Therefore, it is possible to treat neurodegenerative diseases, particularly diseases caused by degeneration of the central nervous system.
  • a neurodegenerative disease is a nerve cell (central nervous system (for example, cranial nerve, spinal nerve), and / or peripheral nerve (for example, autonomic nervous system (for example, sympathetic nerve, parasympathetic nerve)), motor nervous system, sensory nervous system) )
  • central nervous system for example, cranial nerve, spinal nerve
  • peripheral nerve for example, autonomic nervous system (for example, sympathetic nerve, parasympathetic nerve)), motor nervous system, sensory nervous system)
  • any disease that is generally regarded as a neurodegenerative disease may be used.
  • Parkinson's disease Parkinson's syndrome, striatal nigra degeneration, Huntington's disease, chorea-ataxia, progressive Supranuclear paralysis, diffuse Lewy body disease, basal ganglia degeneration, Alzheimer's disease, senile dementia, Pick's disease, frontotemporal lobar dementia, familial dementia, spinocerebellar degeneration (eg olive Bridge cerebellar atrophy, late cerebellar cortical atrophy, familial spinocerebellar ataxia (eg, Maccard Joseph disease, etc.), dentate nucleus erythrocytic Ryukyu atrophy, familial spastic paraplegia, Friedreich Disease), motor neuropathy (eg, amyotrophic lateral sclerosis, familial amyotrophic lateral sclerosis, etc.), demyelinating diseases (eg, multiple sclerosis, multiple sclerosis, acute disseminated) Encephalomyelitis, acute encephalitis, transverse myelitis,
  • the preferred neurodegenerative disease as a subject of the anti-neurodegenerative disease agent of the present invention is, for example, Parkinson's disease, Parkinson's syndrome, Huntington's disease, Alzheimer's disease, senile dementia, spinocerebellar degeneration, amyotrophic lateral sclerosis, Demyelinating diseases (for example, multiple sclerosis), cerebrovascular disorders (for example, stroke, cerebral infarction (for example, cerebral thrombosis, cerebral embolism, etc.), transient cerebral ischemic attacks, cerebral hemorrhage (for example, hypertensive intracerebral hemorrhage) ), Brain tumors, traumatic shock, head injury and / or cerebrospinal trauma (eg, cerebral contusion), cerebrospinal disease (eg, meninges) Inflammation, influenza encephalitis / encephalopathy, Creutzfeldt-Jakob disease, dementia due to AIDS encephalopathy, etc.), diseases derived from central nervous system neurodegeneration such as
  • the anti-neurodegenerative disease agent of the present invention can also treat nerve dysfunction by activating nerve cells, extending neurites, and promoting synapse formation.
  • the target neurological dysfunction may be any neurological dysfunction, such as cognitive dysfunction, consciousness disorder, bilateral quadriplegia, contralateral hemiplegia, alternating hemiplegia. Facial paralysis, sensory impairment, transient blindness (eg, transient cataract), homonymous half-blindness, dizziness, nystagmus, double vision, aphasia, tinnitus, coma and the like. Particularly preferred are those neurological dysfunctions associated with the neurodegenerative diseases.
  • the neurological dysfunction associated with the above-mentioned neurodegenerative diseases for example, neurological dysfunction associated with cerebral infarction varies depending on the site of vascular occlusion, and the symptoms vary depending on the level to be impaired. It can be seen.
  • the presence or absence of neurological dysfunction in cerebral infarction may be determined by various diagnostic tests known in the art for detecting neurological dysfunction. Specific examples of the diagnostic test include, for example, a cognitive function score (Alzheimer's Dissease Assessment-cognitive part; ADAS-cog) used for evaluation of memory and cognitive impairment due to Alzheimer's disease, clinical symptoms, and the like.
  • a cognitive function score Alzheimer's Dissease Assessment-cognitive part
  • the anti-neurodegenerative disease agent of the present invention includes a neuron protective agent, a neuron activator, a neurite extension promoter, a neurite atrophy inhibitor, a Purkinje cell degeneration / dropout inhibitor, and a pathological condition associated with a neurodegenerative disease. It can be advantageously used as a therapeutic agent, a neurological dysfunction therapeutic agent or the like.
  • the treatment of neurodegenerative diseases and neurological dysfunctions as used in the present invention refers to the pathology and dysfunction caused by neurodegeneration in the direction of healing, in addition to so-called treatment, prevention of progression that suppresses progression and stops progression of the disease, Also includes the prevention of the onset of the disease itself.
  • the anti-neurodegenerative disease agent of the present invention can reduce free radicals including hydroxy radicals, it is not limited to tissues such as nerves and blood vessels in the brain, but also in blood vessels and organs other than the brain. It is said to be caused by reperfusion after ischemia, inflammatory diseases (including immune diseases, allergies, tumors, etc.), infections, drugs, radiation, or lipid peroxides generated by physical stimulation. It can be advantageously used as a prophylactic or therapeutic agent for various diseases and conditions.
  • a prophylactic / therapeutic agent for the above-mentioned neurodegenerative diseases but also as a brain protective agent, a brain (nerve cell, vascular endothelial cell) oxidative disorder inhibitor, an ischemic brain disorder inhibitor, a brain Infarct development inhibitor, brain edema inhibitor, delayed neuronal death inhibitor, brain function normalizer, oxidative stress inhibitor, anti-ulcer agent, blood sugar elevation inhibitor, prevention of ocular diseases such as cataract and corneal disorder ⁇
  • Therapeutic / preventive agents for various organ disorders such as impaired skin tissue function, liver damage caused by ischemia, spinal cord injury, arterial and other vascular wall disorders, myocardial and other muscle disorders, and tubulointerstitial disorders Disorder prevention / treatment agent, anti
  • the anti-neurodegenerative disease agent of the present invention includes an amyloid ⁇ peptide aggregation inhibitor, an amyloid ⁇ peptide injury inhibitor, a cholinesterase activity inhibitor, a serine / threonine kinase (Akt) activator, a phosphatidylinositol (3,4).
  • Akt serine / threonine kinase
  • phosphatidylinositol 3,4
  • Triphosphate kinase (PI3K) -serine / threonine kinase (Akt) cascade activator, cyclic AMP concentration increase promoter, SAPK / JNK phosphorylation inhibitor, etc. are also optional.
  • NK-4 a compound represented by Chemical Formula 2 (manufactured by Hayashibara Biochemical Laboratories, Inc., “NK-4”) was used as a test sample. Since NK-4 is hardly soluble in water, it is dissolved in DMSO (SIGMA, product number “D8418”) at a concentration of 5 mg / ml, and then membrane filtered (Millipore, product name “Millex-LG SLLG025SS”, DMSO resistant membrane was used), and further diluted with Dulbecco's MEM medium (sold by Nissui Pharmaceutical Co., Ltd., hereinafter abbreviated as “D-MEM medium”), and subjected to the test.
  • D-MEM medium Dulbecco's MEM medium
  • NGF Neve Growth Factor
  • PC12-HS cells PC-12 cells derived from rat adrenal pheochromocytoma
  • FBS fetal bovine serum
  • Cells used in the test were detached by a conventional method using a 0.25% by mass trypsin solution, diluted with 10% by volume FBS-added D-MEM medium, and coated with a collagen-coated 96-well plate (available from Falcon, trade name). “Micro test plate / cell culture, flat bottom”) was seeded at 5 ⁇ 10 3 cells / 100 ⁇ l / well. After 24 hours, the culture supernatant was removed, diluted with D-MEM medium containing no FBS, and 100 ⁇ l / well of NK-4 adjusted to twice the final concentration shown in Table 1 was added for 3 days. Cultured.
  • PC12-HS cells cultured in the same manner as above were diluted with 10% by volume FBS-added D-MEM medium, seeded at 2 ⁇ 10 4 cells / 100 ⁇ l / well in a collagen-coated 96-well plate, and cultured for 24 hours. did. Then, dilute 800 ⁇ M hydrogen peroxide solution (available from Wako Pure Chemical Industries, Ltd.) with 50 ⁇ l / well (final concentration 200 ⁇ M) and 10% FBS-added D-MEM medium to a concentration 4 times the final concentration shown in Table 1.
  • NK-4 50 ⁇ l / well of each of NK-4 was added simultaneously (final concentration of NK-4 from 5 ng to 50,000 ng / ml), cultured in an incubator for 2 hours, and then 25 vol% glutaraldehyde (Wako Pure Chemical Industries, Ltd.). The cells were fixed by adding 20 ⁇ l / well (final concentration 20% by volume). 0.05 ⁇ m methylene blue (available from Wako Pure Chemical Industries, Ltd.) was added at 100 ⁇ l / well, and the absorbance of each well was measured by a conventional method using a die-up take method. As a control, culture was carried out in the same manner except that hydrogen peroxide and NK-4 were not added, and methylene blue was added to measure the absorbance. The relative value when the number of cells of the control (absorbance) is 100 (%) is determined and shown in Table 1 as the cell viability (%) of each well.
  • ⁇ Influence of NK-4 on cell injury by amyloid ⁇ fragment Peptide fragments having an amino acid sequence corresponding to the 25th to 35th amino acids from the amino terminus of amyloid ⁇ peptide (human origin), which is considered to be one of the main causes of neuronal cell death in Alzheimer's disease (sold by AnaSpec, hereinafter “Amyloid ⁇ Fragment ”) (a peptide having the amino acid sequence of SEQ ID NO: 1 in the sequence listing) was diluted with phosphate buffered saline (PBS) to a concentration of 2 mM, and 6 hours at 37 ° C before use. Aged and aggregated fragments were used to increase cytotoxicity.
  • PBS phosphate buffered saline
  • PC12-HS cells cultured in the same manner as above were diluted with 10% by volume FBS-added D-MEM medium, and seeded in a 96-well plate coated with collagen at 5 ⁇ 10 3 cells / 100 ⁇ l / well. After culturing for 24 hours, the supernatant was removed, and 50 ⁇ l / well of amyloid ⁇ fragment solution diluted with 10% by volume FBS-added D-MEM medium (final concentration of amyloid ⁇ fragment 50 ⁇ M) and NK-4 solution were 50 ⁇ l / well. (Final concentration 40 to 5000 ng / ml) was added and cultured for 3 days.
  • NK-4 has a difference in effective concentration for each cytotoxic factor against nutrient starvation, hydrogen peroxide damage, and amyloid ⁇ fragment damage to PC12-HS cells. However, it has been found to have a protective effect on nerve cells. Comparing the concentrations of NK-4 that exert the protective action against the three types of cytotoxic factors shown in Table 1, the protective action against the damage of the amyloid ⁇ fragment is exerted from 40 ng / ml, whereas the nutrient starvation It was found that a concentration of 500 ng / ml was required for injury and a concentration of 5,000 ng / ml was required for hydrogen peroxide injury.
  • NK-4 aqueous solution having a concentration of 50,000 ng / ml or less does not have the ability to erase hydrogen peroxide.
  • the cytoprotective action against cell damage by hydrogen peroxide in this test system is not due to the action of directly removing hydrogen peroxide, but acts on the cell side, Judged to suppress death.
  • the occupancy ratio of the cells having undergone apoptosis calculated from Hoechst staining image was 72% when amyloid ⁇ fragment was added (NK-4 concentration 0 ng / ml), whereas apoptosis was promoted.
  • -4 200 ng / ml was added, the occupancy was 13%, which was close to the occupancy (5%) of apoptotic cells in the control, and NK-4 was reduced by amyloid ⁇ fragment. It was confirmed to suppress the induced apoptosis.
  • specific data are not shown, cell aggregation and cell death were observed by addition of amyloid ⁇ fragment in phase contrast microscopy, whereas cell aggregation and cell death were observed by addition of NK-4.
  • NK-4 can be used as a neuroprotective agent or neurotrophic factor because it has neurotrophic factor activity because it has a neuroprotective action against nutrient starvation injury. ing.
  • NK-4 protects cells against a plurality of cytotoxic factors and suppresses cell death. Therefore, NK-4 has neurodegeneration inhibitory activity and cytotoxicity including amyloid ⁇ peptide. It shows that it can be used as an effective therapeutic agent for human neurodegenerative diseases represented by factors such as Alzheimer's disease. It also shows that NK-4 can be used as an apoptosis inhibitor.
  • NGF nerve growth factor
  • NK-4 inhibits the production of phosphatidylinositol (3,4,5) triphosphate by inhibiting phosphatidylinositol (3,4,5) triphosphate kinase (PI3K).
  • LY294002 Vlahos C. et al., “Journal of Biological Chemistry”, Vol. 269, Suppresses and ultimately suppresses the activation of serine / threonine kinase (Akt), which plays a major role in cell survival and proliferation. 5241-5248 (1994)
  • Akt serine / threonine kinase
  • NK-4 has an effect of inducing an increase in intracellular cyclic AMP (adenosine monophosphate) concentration.
  • SAPK Stress activated protein kinase
  • JNK c-Jun N-terminal Kinase phosphorylation
  • NK-4 has a neurodegenerative inhibitory effect
  • cerebellar degeneration ataxia (hereinafter referred to as a suitable model animal for human neurodegenerative diseases such as spinocerebellar degeneration)
  • the hamster (hereinafter referred to as “cerebellar ataxia hamster”) was used to examine the effects of NK-4 administration on its behavior and brain tissue.
  • test group 1 hamsters were administered with 10 ml / kg / day of PBS before the onset of cerebellar ataxia (3 weeks of age).
  • the hamsters in test groups 2 to 4 were administered NK-4 at 20 ⁇ g / kg, 100 ⁇ g / kg, or 500 ⁇ g / kg / day before the onset of cerebellar ataxia (3 weeks of age).
  • the hamsters in Test Group 5 had insulin-like trophic factor-1 known as a neuronal trophic factor-1 (sold by Assaypro, trade name “IGF-1, human”) from the beginning of cerebellar ataxia (3 weeks old) Hereinafter, abbreviated as “IGF-1”) was administered at 25 ⁇ g / kg / day. These administration components were administered intraperitoneally once daily until 10 weeks of age.
  • the degree of cerebellar ataxia and the effect of NK-4 on improving the symptoms were evaluated by using the rotarod test and slope endurance test described later once a week, with the improvement of hamster motor coordination as an index. Furthermore, at the age of 10 weeks, after measuring the number of hamster falls, the brain was removed and histologically evaluated, and the glutamic acid concentration in blood and cerebrospinal fluid (CSF) was also measured. As test group 6, 5 normal hamsters of the same age as the cerebellar ataxia hamsters used in test groups 1 to 5 were subjected to intraperitoneal administration of 10 ml / kg / day of PBS once daily until 10 weeks of age. Then, the same test as the cerebellar ataxia hamster was conducted.
  • CSF cerebrospinal fluid
  • ⁇ Rotarod test> The hamster walked with the rotation of the rotarod and used the duration of the exercise to stay on the rotarod as an indicator of motor coordination. That is, a hamster was placed on a rotarod apparatus (manufactured by Hayashibara Biochemical Laboratories Co., Ltd., rotarod diameter 60 mm) rotating at a constant speed (6 rpm), and the time until it dropped from the rotarod was measured (Fernandez et al., “Proc. Natl. Acad. Sci. USA, 95, 1253-1258 (1998)). The test is conducted 6 times for one hamster, and the first 5 times is a preliminary motion test for acclimatization to the rotational motion.
  • the cerebrum and cerebellum were photographed from above at a certain height with a digital camera, and the respective sagittal and horizontal diameters were measured.
  • Sagittal length 2 ⁇ horizontal length ⁇ 0.5 was calculated as cerebrum volume and cerebellum volume, respectively, and the average of 5 animals in each group was determined. The results are shown in Table 5.
  • cerebellar ataxia hamster used in this test is known to have a reduced cell density in addition to Purkinje cells, which are inhibitory neurons, and granule cells, which are excitatory neurons. Therefore, cerebellar slices cut in the sagittal direction were stained with hematoxylin and eosin by a conventional method, and microscopically measured to determine the total number of Purkinje cells in the Purkinje cell layer (lobules I to X) and the number of granule cells per unit area. In addition, the number of individuals with demyelination in the cerebellar white matter was confirmed. The results are also shown in Table 5. In addition, since the cerebral volume did not recognize a significant difference between each test group, Table 5 shows only the calculation result of the cerebellum volume.
  • GABA ⁇ -aminobutyric acid
  • the drop time of a 4-week-old normal hamster was 180 seconds or more, whereas Test Group 1 (using the same 4-week-old cerebellar ataxia hamster) (The fall time of the PBS administration group was 108 ⁇ 10 seconds, and a significant shortening of the fall time was already observed as compared with normal hamsters. At the age of 10 weeks, it was not possible to stay on the rotarod and it immediately dropped (0 seconds). In contrast, in the hamsters administered with NK-4, the reduction of the fall time was observed in a dose-dependent manner from the first week (4 weeks of age) after the start of administration, and in test group 3 (100 ⁇ g / kg / body weight).
  • test group 4 (500 ⁇ g / kg ⁇ body weight), a significant effect of reducing the drop time was observed compared to test group 1. This inhibitory effect lasted until the end of the study at 10 weeks of age (7 weeks of administration). Even in Test Group 2 (20 ⁇ g / kg / body weight), after 2 weeks (5 weeks old) after the start of NK-4 administration, the effect of reducing the drop time was observed more than in Test Group 1, but Test Group 3 and Compared with test group 4, the effect was weak. In addition, when IGF-1 which is considered to be effective in the treatment of motor neurodegenerative diseases was administered (Test Group 5), the effect of suppressing the fall time was hardly observed.
  • test group 6 normal hamster
  • test group 1 using cerebellar ataxia hamsters.
  • test group 5 IGF-1 administration
  • 44.4 ⁇ 0.2 degrees at 4 weeks of age which is a significant slope durability inclination angle compared to 40.6 ⁇ 0.3 degrees in test group 1
  • the slope durability inclination angle decreased as in Test Group 1, and no significant improvement was observed after 5 weeks of age.
  • test groups 2 to 4 In contrast, in test groups 2 to 4 (NK-4 administration), no decrease in endurance tilt angle was observed throughout the test period at any dose used in the test, and a high slope endurance tilt angle decrease suppression effect was observed. It was.
  • the slope endurance slopes of the test groups 2 to 4 (NK-4 administration) at 10 weeks of age are 45.8 ⁇ 0.6, 51.6 ⁇ 0.6, and 51.8 ⁇ 0.4 degrees, respectively. Both were significantly higher than those in Test Group 1 (PBS administration) and Test Group 5 (IGF-1 administration).
  • test group 6 As is apparent from the results in Table 5, no fall was observed in the 10-week-old normal hamster (test group 6), whereas in test group 1 (PBS administration), 12.8 ⁇ 0.5 times A fall of / min was observed. In contrast, in test group 5 (IGF-1 administration), 11.4 ⁇ 0.4 times / min was slightly decreased compared to test group 1, but a significant fall-reducing effect was observed. I could't. In contrast, in test groups 2 to 4 (NK-4 administration), 4.0 ⁇ 1.0, 1.6 ⁇ 0.9, and 1.2 ⁇ 0.8 times / min, respectively, There was also a significant decrease in the number of falls in the group.
  • test group 1 PBS administration
  • test group 6 IGF-1 administration
  • test group 5 IGF-1 administration
  • a significant cerebellar atrophy suppression effect was observed as compared to test group 1, which was 77.6 ⁇ 6.1 (mm 3 ).
  • a dose-dependent cerebellar atrophy suppression effect was also observed in the NK-4 administration group, and 76.0 ⁇ 8.2, 77.0 ⁇ 2.8, 80 in the 20, 100, and 500 ⁇ g / kg administration groups, respectively. 5 ⁇ 10.8 mm 3 (all significant at P ⁇ 0.05).
  • NK-4 improves motor coordination of cerebellar ataxia hamsters, effectively suppresses cerebellar atrophy, and its effect is superior to IGF-1. .
  • the granule cell density in the granule cell layer of the cerebellar cortex is 480 ⁇ 6 cells / 20,000 ⁇ m 2 for 10-week-old normal hamsters (test group 6).
  • test group 6 when PBS was administered to cerebellar ataxia hamsters (Test Group 1), the number decreased significantly to 380 ⁇ 4 / 20,000 ⁇ m 2 .
  • the granule cell density in test group 5 (IGF-1 administration) was 371 ⁇ 11 cells / 20,000 ⁇ m 2, which was not different from test group 1.
  • test groups 2 to 4 (NK-4 administration), 408 ⁇ 8, 419 ⁇ 6, 436 ⁇ 7 cells / 20,000 ⁇ m 2 were obtained, respectively. Admitted.
  • test group 1 In addition, although specific data are not shown, in microscopic observation of the cerebellar parenchyma, in the test groups 1 and 5, granule cell atrophy and degeneration were significant, whereas in the test groups 2 to 4, granule cells were observed. It was confirmed that atrophy and degeneration were suppressed. Furthermore, when PBS was administered to a cerebellar ataxia hamster (test group 1), demyelination of cerebellar white matter was observed in all individuals (5 of 5 animals), whereas in the NK-4 administration group, test groups 2 to 4 (NK-4 administration), demyelination was only observed in 4 out of 5 animals, 1 out of 5 animals, and 0 out of 5 animals.
  • NK-4 is also useful as an inhibitor of cerebellar white matter demyelination, ie, an inhibitor of Purkinje cell degeneration and loss, and an atrophy inhibitor of neuronal cell processes such as Purkinje cells. It also tells us that it can be used as an extension accelerator.
  • NK-4 administered intraperitoneally acted on cerebral neurons and suppressed cerebellar ataxia.
  • NK-4 suppressed the decrease in glutamate content in CSF of cerebellar ataxic hamsters
  • NK-4 suppressed the decrease in neuronal function associated with neurodegeneration through the activation of neurons. And it shows that it can control the decline of motor ability and learning ability.
  • NK-4 is useful as a systemically administrable agent for human neurodegeneration and various pathological and clinical symptoms associated therewith.
  • the body weight of the cerebellar ataxia hamster was measured once a week until the end of the test (10 weeks of age) and the average of each group was determined.
  • PBS, NK-4, IGF-1 administration group Since no significant difference was observed, NK-4 was judged to be a highly safe compound even when continuously administered to a living body for a long period of time.
  • NK-4 has a protective action against cytotoxic factors, an inhibitory effect on neurodegeneration, an inhibitory effect on Purkinje cell reduction that causes cerebellar ataxia, and an inhibitory action on neuronal cell loss. It was examined whether or not a dye compound other than 4 (hereinafter sometimes simply referred to as “compound”) has the same effect.
  • ⁇ Test sample> Since 239 types of compounds shown in Table 6 are hardly soluble in water, they were dissolved in DMSO (SIGMA, catalog number “D8418”) at a concentration of 5 mg / ml, as in the case of NK-4. Thereafter, the membrane was filtered with Millex-LG (sold by Millipore, product number “LLG025SS”, DMSO resistant), and stored at 25 ° C. protected from light. At the time of use, a test sample was prepared by diluting 200-fold or more with 10% by volume FBS-added D-MEM medium (Nissui Pharmaceutical), and subjected to the test. These compounds were all synthesized by Hayashibara Biochemical Laboratories.
  • ⁇ Evaluation method A Evaluation method of nerve cell proliferation promoting action>
  • PC12-HS cells were diluted with D-MEM medium supplemented with 10% by volume FBS to a pre-collagen-coated 96-well microplate at 5 ⁇ 10 3 cells / well, and 100 ⁇ l / well. Sowing. After 24 hours, each test sample diluted with 10 volume% FBS-added D-MEM medium and adjusted to 100 ng / ml was added at 100 ⁇ l / well, and cultured in a 37 ° C., 5 volume% CO 2 incubator for 3 days.
  • ⁇ Evaluation method B Evaluation method of neurite extension action>
  • PC12-HS cells were diluted with D-MEM medium supplemented with 10% by volume FBS to a 96-well microplate previously coated with collagen at 5 ⁇ 10 3 cells / well. And 100 ⁇ l / well. 24 hours later, each test sample was diluted with 10% by volume FBS-added D-MEM medium and adjusted to 400 ng / ml, 50 ⁇ l / well, and 20 ng / ml NGF (available from Chemicon, mouse origin, final concentration 5 ng / ml) 10 50% l / well of D-MEM medium supplemented with volume% FBS was added and cultured for 3 days.
  • the cells were fixed with 10% by volume glutaraldehyde for 20 minutes at room temperature.
  • PC12-HS cells cultured for 3 days only in 10% by volume FBS-added D-MEM medium were fixed with glutaraldehyde.
  • the fixed cells were observed under a microscope to evaluate the presence or absence of neurite outgrowth, and when the neurite outgrowth rate was 30% or more, it was determined to be equal to or better than NK-4 ( ⁇ ).
  • the neurite outgrowth rate (%) is the same by observing cells under a microscope at a magnification including about 100 cells in one field, counting the number of cells having neurites more than twice the cell body, Dividing by the total number of cells in the field of view and multiplying by 100 was obtained. Further, when only NGF was added to this experimental system (5 ng / ml), the neurite extension rate was about 5%. The results are also shown in Table 6.
  • NK-19 compound represented by chemical formula 4
  • NK-53 compound represented by chemical formula 5
  • NK-100 compound represented by chemical formula 6
  • NK-528 expressed by chemical formula 7
  • Compound Compound
  • NK-557 compound represented by chemical formula 8
  • NK-1516 compound represented by chemical formula 9
  • NK-557 (compound represented by Chemical Formula 8), and NK-1516 (compound represented by Chemical Formula 9) have a strong cell growth promoting action and neurite outgrowth promoting action. It is useful as a neurodegenerative disease agent. Furthermore, these compounds, particularly NK-4, NK-19, NK-53, NK-100, NK-528, NK-557, NK-1516, are human neurodegenerative diseases and associated pathological and neurological dysfunctions. It can be used as a therapeutic agent. In addition, Table 6 shows that a compound having an action of suppressing cell damage caused by amyloid ⁇ fragment is useful as an apoptosis inhibitor.
  • NK-19, NK-53, NK-100, NK-528, NK-557, and NK-1516 were confirmed to have the effect of inhibiting cell damage caused by amyloid ⁇ fragment in the same manner as NK-4.
  • the effect of the concentration on cell damage by amyloid ⁇ fragment was examined. That is, except that the above 6 kinds of compounds and NK-4 were used as test preparations and each compound was added to the wells seeded with PC12-HS cells so as to have final concentrations shown in Table 7, the experiment 3 Under the same conditions as in Evaluation Method C, the inhibitory effect of these compounds on cytotoxicity by amyloid ⁇ fragment was evaluated. The results are shown in Table 7 as cell viability (%).
  • NK-19, NK-53, NK-100, and NK-557 were at a lower concentration than NK-4, and showed high inhibitory activity against cell damage caused by amyloid ⁇ fragment.
  • NK-53 showed the highest activity at the lowest concentration, and the cell damage by amyloid ⁇ fragment was almost completely suppressed at a concentration of 12.5 ng / ml (cell survival rate 115 ⁇ 16%).
  • the injury inhibition rate was highest at a concentration of 50 ng / ml, which was 102 ⁇ 27%, 88 ⁇ 12%, and 114 ⁇ 9%, respectively.
  • NK-53 was found to have a high inhibitory activity of 111 ⁇ 9% even at a concentration of 50 ng / ml.
  • NK-4 had the highest injury inhibition rate of 122 ⁇ 32% at a concentration of 200 ng / ml, whereas NK-19, NK-53, NK-100, and NK-557 had a cytoprotective effect. It was confirmed that there was an optimal concentration for the cytotoxic effect of these compounds, and the optimal concentrations of NK-19, NK-53, NK-100, and NK-557 are NK- It was found to be lower than 4.
  • NK-528 and NK-1516 did not show a higher cytotoxic effect than NK-4. From the above results, it is shown that NK-19, NK-53, NK-100, and NK-557 may exhibit superior effects at lower concentrations than NK-4 for neurodegenerative diseases such as Alzheimer's disease. It was done. In addition, NK-19 and NK-100, which showed a high cytotoxicity-inhibiting activity at a lower concentration than NK-4, had a higher molecular weight than the others, and NK19 and NK-53 bound to nitrogen in the thiazole ring. Since the number of carbon atoms in the side chain alkyl group is as large as 7 (other carbon number is 2), it was judged that strong activity was expressed because of high lipid solubility and high cell membrane permeability.
  • NK-4, NK-19, NK-53, NK-100 and NK-4, NK-19 which were confirmed to have a neurite outgrowth promoting action among the compounds in which the protective action against the cytotoxicity of amyloid ⁇ fragment was recognized.
  • NK-557 as a test sample, a test for examining the effect on aggregation of amyloid ⁇ peptide, which is a suitable model for developing a therapeutic agent for human Alzheimer's disease, was performed as follows.
  • each test sample was dissolved in DMSO (SIGMA, catalog number “D8418”) at a concentration of 5 mg / ml, and then membrane-filtered with Millex-LG (Millipore, product number “LLG025SS”, DMSO resistant). Using Tris-HCl buffer, the test sample solution was prepared to a concentration of 200 nM.
  • test sample When the test sample inhibits amyloid ⁇ peptide aggregation, the thioflavin-T fluorescence decreases.
  • the effect of the test sample on amyloid ⁇ peptide aggregation was examined by this method. That is, a human amyloid ⁇ peptide having an amino acid sequence consisting of 40 amino acids represented by SEQ ID NO: 2 in the Sequence Listing (sold by Ana Spec) was used after dissolving in sterile distilled water at a concentration of 400 ⁇ M. The test sample solution was diluted with Tris-HCl buffer.
  • NK-4, NK-19, NK-53, NK-100 and NK-557 used in the test suppressed aggregation of amyloid ⁇ peptide
  • NK-19, NK- 53, NK-100 and NK-557 all showed stronger inhibitory activity than NK-4.
  • NK-53, NK-100 and NK-557 suppressed amyloid ⁇ peptide aggregation by 95% or more
  • NK-100 almost completely suppressed aggregation by 99%.
  • a compound having an action of suppressing cell damage caused by amyloid ⁇ fragment such as NK-4, NK-19, NK-53, NK-100 and NK-557 has an action of suppressing aggregation of amyloid ⁇ peptide. Therefore, it shows that it is useful not only as a therapeutic agent for Alzheimer's disease but also as a preventive agent.
  • NK-4 and NK-100 among the compounds added to the medium showed a particularly strong cell growth promoting action.
  • the optimal concentration was observed for the cell growth promoting action by these compounds.
  • NK-19, NK-53 and NK-100 were 50 ng / ml, whereas NK-4 and NK-557 were 200 ng / ml.
  • the neurite extension promoting action increased depending on the concentration of each compound, and the strongest neurite extension promoting action was observed with NK-100.
  • a midline cervical incision was made and the right carotid bifurcation was reached, paying attention to the preservation of the vagus nerve.
  • the common carotid artery and the external carotid artery were peeled from the surrounding connective tissue, centering on the right carotid bifurcation, and ligated with 6-0 nylon thread (sold by Alfresa Pharma Co., Ltd., trade name “Nesscoacher”). Next, a 6-0 nylon thread was applied to the internal carotid artery to prepare for fixation after embolization.
  • the common carotid artery was incised, and an embolus (Doccol) made of 4-0 nylon thread coated with silicon at the tip was inserted into the internal carotid artery about 16 mm and fixed to the common carotid artery with a clip.
  • Doccol embolus
  • the tip portion of the embolus that is silicon-coated enters the anterior cerebral artery beyond the middle cerebral artery bifurcation and closes the middle cerebral artery entrance.
  • the cerebral artery is occluded for 2 hours on a thermal pad at 37 ° C., then the inserted embolus is removed, blood flow is resumed (reperfusion), and then bleeding from the common carotid artery incision is prevented. Therefore, the internal carotid artery was ligated near the carotid bifurcation. Since the right common carotid artery is ligated in this model, the blood flow is resumed from the left inner diameter artery, the vertebral artery, and the basilar artery via the anterior / posterior traffic artery.
  • NK-4, NK-19, NK-53, NK-100 and NK-557 used in the test were each dissolved in DMSO (SIGMA, product number “D8418”) at a concentration of 5 mg / ml, and then the membrane. Filtration (sold by Millipore, trade name “Millex-LG SLLG025SS”, using DMSO resistant membrane) was performed. Each compound was dissolved in PBS to 25 ng / ml at the time of use, and any one of them was administered to 5 or 7 rats in 5 groups each 1 hour after middle cerebral artery occlusion and blood reperfusion.
  • DMSO product number “D8418”
  • test groups 1 to 5 were administered into the tail vein (4 ml / kg ⁇ body weight, compound dose of 100 ⁇ g / kg ⁇ body weight) (test groups 1 to 5). 24 hours after resumption of blood flow, behavioral and histological evaluations were performed based on the method described below. Of the remaining 2 groups, 5 mice per group were treated with the same procedure as in test groups 1 to 5 as control group 1, followed by PBS containing no compound at 4 ml / kg ⁇ body weight, middle cerebral artery occlusion 1 It was administered via the tail vein after time and at the time of blood reperfusion.
  • control group 2 In the remaining 6 animals in 1 group, as control group 2, after ligation of the common neck, external neck, and internal carotid artery, sham operation was resumed without inserting an embolus near the middle cerebral artery ( (Sham operation) was performed.
  • PBS containing no compound was also administered into the tail vein at 4 ml / kg / body weight, the common neck, the external neck, and the internal carotid artery 1 hour after ligation and at the time of blood reperfusion.
  • behavioral and histological evaluations similar to those of test groups 1 to 5 were performed.
  • TTC 2,3,5-triphenyltetrazole chloride
  • the area of the cerebral infarction site stained with TTC was analyzed using image analysis free software (trade name “Scion Image”, sold by Scion), and the volume of the infarct site and the entire brain was calculated.
  • the volume of the infarcted area was divided by the total volume of the brain, multiplied by 100, and the ratio (%) of the infarcted area to the entire brain was calculated.
  • the relative value of the ratio of the cerebral infarction site of the rats of the test groups 1 to 5 to the entire brain when the ratio of the cerebral infarction site of the rats of the control group 1 to 100% is calculated, The size of cerebral infarction (%).
  • the results are also shown in Table 12.
  • NK-4, NK-19, NK-53, NK-100 and NK-557 were compared with those of rats (control group 1) that had cerebral ischemia. In either case (test groups 1 to 5), the site of cerebral infarction was small in any case, and in NK-4, NK-19, and NK-53 (test groups 1 to 3) A significant reduction in the cerebral infarction site was observed. When the strength of the effect was compared, the strongest improvement was observed in both the behavioral score and the inhibition rate of the cerebral infarction site when NK-19 was administered. This result shows that NK-4, NK-19, NK-53, NK-100 and NK-557 have a therapeutic effect on neurodegeneration caused by ischemia and the subsequent resumption of blood flow and the associated neurological dysfunction. Telling you to have.
  • AchE inhibitors such as donepezil have been clinically applied to Alzheimer-type dementia. It has been reported that AchE inhibitors activate the central cholinergic nervous system and improve cognitive function even in ischemic dementia. Therefore, NK-4, NK-19, NK-53, NK-100, and NK-557, which were confirmed to have an effect of improving cerebral infarction and the accompanying neurological dysfunction in rats with cerebral ischemia in Experiment 7, A test was conducted to confirm whether there was an AchE inhibitory effect.
  • NK-4, NK-19, NK-53, NK-100 and NK-557 dissolved in DMSO were each diluted with a phosphate buffer, and a compound having a concentration 10 times that shown in Table 13 was added.
  • a solution was prepared to provide a test sample solution.
  • PC12-HS cells cultured in the same manner as in Experiment 1 were collected, and 5 volumes (volume) of 10 mM Tris-HCl buffer solution (1M NaCl, 50 mM MgCl 2 , 1% Triton X-100, pH 7.2) was added. After homogenizing uniformly by a conventional method, the mixture was centrifuged (10,000 g) at 4 ° C.
  • acetylcholinesterase AchE
  • a solution containing acetylcholinesterase AchE
  • 30 ⁇ l of 50 mM phosphate buffer (pH 8.0) 10 ⁇ l of test sample solution, and 10 ⁇ l of AchE-containing solution were added.
  • reaction substrate solution 50 ⁇ l of a phosphate buffer containing 0.5 mM acetylthiocholine iodide (sold by Wako Pure Chemical Industries, Ltd.) and 1 mM 2-nitrobenzoic acid (sold by Wako Pure Chemical Industries, Ltd.) was added. After an enzyme reaction for 30 minutes in a 37 ° C. incubator, the absorbance (A R ) at 405 nm was measured with a plate reader. Further, using 10 ⁇ l of phosphate buffer instead of the AchE-containing solution, the enzyme reaction was carried out in a 37 ° C. incubator for 30 minutes in the same manner as described above, and the absorbance (A U ) was measured.
  • NK-100 showed the highest AchE inhibition in the low concentration range, and showed a significant AchE inhibitory effect when the concentration of NK-100 was 0.78 ⁇ g / ml or more.
  • NK-4 having a structure similar to that of NK-100 was significantly inhibited at a concentration of 3.13 ⁇ g / ml or more.
  • the IC 50 of these compounds was 3.3 for NK-4 and 11.8 ⁇ g / ml for NK-100.
  • NK-19 and NK-53 had weak AchE inhibitory activity, and NK-19 and NK-53 showed a significant decrease in activity only at a concentration of 25 ⁇ g / ml.
  • NK-557 showed almost no AchE inhibitory activity at concentrations up to 25 ⁇ g / ml.
  • the four compounds NK-4, NK-19, NK-53, and NK-100 also activated the cholinergic nervous system by the inhibitory action of AchE, and caused Alzheimer-type dementia and ischemic cognition. The possibility of improving the symptoms was shown.
  • galantamine which is clinically used as a therapeutic agent for Alzheimer's disease, shows the same level of AchE inhibitory effect at a lower concentration than NK-4 (galantamine has an IC 50 of 442 ⁇ g / ml).
  • these compounds have also been reported to suppress cell death via the PI3K-Akt cascade similar to NK-4, but NK-4, NK-19, NK-53, NK-100 and NK-557. The effect is obtained at several hundred ng / ml, whereas donepezil, galantamine, and tacrine differ in the order of the concentration of the compound necessary to obtain the same effect.
  • NK-4, NK-19, NK-53, NK-100, and NK-557 are considered to be existing Alzheimer's in view of these facts. It shows that it can be used as an anti-neurodegenerative disease agent having a mechanism of action different from that of a disease treatment drug.
  • Peroxy radical is a kind of in vivo lipid peroxide generated by the reaction of unsaturated fatty acid and hydroxy radical.
  • the brain with high lipid content is greatly affected by peroxy radical.
  • Peroxy radical scavenging activity caused by overheating of azobis (2-amidinopropane) dihydrochloride (AAPH) is a model of biologically derived radicals that are eliminated by antioxidant components. Therefore, a test for confirming whether NK-4, NK-19, NK-53, NK-100, and NK-557 have peroxy radical scavenging ability was conducted by using ESR to erase peroxy radicals generated from AAPH. This was done by measuring.
  • NK-4, NK-19, NK-53, NK-100 and NK-557 dissolved in DMSO are each diluted with purified water to prepare a solution containing a compound having a concentration 3 times the concentration shown in Table 14.
  • a test sample solution was obtained.
  • edaravone a brain protective agent with the action mechanism of free radical scavenging (sold by Mitsubishi Tanabe Pharma Corporation, trade name “Radicut”, active ingredient: 3-methyl-1-phenyl-2-pyrazolin-5-one) was diluted with purified water to a concentration 3 times that shown in Table 14 and used.
  • NK-4, NK-19, NK-53, NK-100 and NK-557 dissolved in DMSO are each diluted with 0.1 M phosphate buffer, and the concentration is a compound having a concentration three times that shown in Table 15.
  • a solution containing was prepared as a test sample solution.
  • ESR measurement method The reaction solution for hydroxy radical measurement or peroxy radical measurement is placed in a flat quartz cell for ESR and set in an electron spin resonance (ESR) apparatus (trade name “Free radical Monitor JES-FR30” sold by JEOL Ltd.). ESR was measured according to Instead of the test sample solution, purified water is used in the case of hydroxy radical measurement, and 0.1 M phosphate buffer is mixed in the case of peroxy radical measurement, and the test sample solution is added and reacted in the same manner. React and set the value when measuring ESR as 100, determine the relative strength when the test sample solution was added and reacted, and as the residual rate of hydroxy radicals (%) and the residual rate of peroxy radicals (%), They are shown in Tables 14 and 15, respectively.
  • NK-4, NK-19, NK-53, NK-100 and NK-557 are more than commercially available free radical scavengers for brain protection. It was also revealed that it has excellent hydroxy radical scavenging ability. Further, IC 50 of hydroxy radical scavenging ability of NK-9694 and NK-150 measured by the same method were 3.4 ⁇ g / ml and 1.8 ⁇ g / ml, respectively.
  • NK-557 and NK-100 peroxy radicals were significantly eliminated at concentrations of 25 ⁇ g / ml and 50 ⁇ g / ml or more, respectively. From the above results, all five compounds used in the test have strong hydroxy radical scavenging ability, and the three compounds NK-4, NK-19, and NK-53 are peroxyl even at relatively low concentrations. Since it also exhibits radical scavenging activity, it was considered that free radical scavenging ability such as hydroxy radicals and peroxy radicals was involved in one of the main action mechanisms of infarct reduction effect in model rats with cerebral infarction. .
  • the NK-4, NK-19, and NK-53 administration groups showed higher infarct reduction effects than the NK-557 and NK-100 administration groups.
  • edaravone used as a positive control has free radical scavenging ability (free radical scavenger), suppresses the generation of hydroxy radicals in the brain after blood reperfusion in a rat cerebral ischemia model, and cerebral infarction It is known to have nest development, blood flow reduction inhibitory effect around the cerebral infarction, brain edema and delayed neuronal cell death (for example, “The Japanese Pharmacology Journal” 119, 301-308 (2002)).
  • the anti-neurodegenerative disease agent of the present invention has a stronger hydroxy radical scavenging ability than edaravone, and it has been found that it has a cerebral infarction progression inhibitory action and a nerve cell death inhibitory action.
  • Japanese Patent Application Laid-Open No. 2008-37753 Japanese Patent Application Laid-Open No. 2008-37753
  • protein kinase stimulator Japanese Patent Application Laid-Open No. 2004-339214
  • preventive / therapeutic agent for mitochondrial encephalomyopathy Japanese Patent Laid-Open No. 2005-89456
  • arterial occlusion / stenosis prevention / treatment Agent Japanese Patent Laid-Open No. 2005-162749
  • blood brain barrier breakdown inhibitor International Publication WO04 / 63167 pamphlet
  • drug dependence treatment agent Japanese Patent Laid-Open No. 2008-247813
  • apoptosis inhibitor Japanese Patent Laid-Open No.
  • the anti-neurodegenerative disease agent of the present invention can be advantageously used as a prophylactic / therapeutic agent for these diseases having an effect equivalent to or better than that of edaravone. It tells you what you can do.
  • NK-19 Neurotrophic factor-like activity of NK-19 analog
  • the number of carbon atoms in the alkyl group of the side chain (R 7 to R 9) is 1 to 12
  • counter anion I - or Cl - the a is 12 kinds of compound synthesis (Synthesis by Hayashibara Biochemical Laboratories Co., Ltd.)
  • the strength of the cell growth promoting action and neurite extension promoting action on PC12-HS cells was examined.
  • NK-19 analogs including NK-19 shown in Table 16 were dissolved in DMSO to a concentration of 5 mg / ml. This solution was diluted with 10% by volume FBS-added D-MEM medium to prepare a test sample solution having a compound concentration of 100 ng / ml or 2 ⁇ g / ml, respectively. Moreover, the NK-24 and NK-19 is the counter anion I - Cl from - compounds instead (NK-56 and NK-53) was also dissolved at a 5 mg / ml in DMSO. These solutions were diluted with 10% by volume FBS-added D-MEM medium to prepare test sample solutions by diluting the compound concentration to 100 ng / ml or 2 ⁇ g / ml, respectively.
  • the cells were cultured for 3 days only in D-MEM medium supplemented with 10% by volume FBS, and the cells were fixed with glutaraldehyde. The fixed cells were observed under a microscope, and the presence or absence of neurite extension was evaluated by the same method as in Experiment 3. The results are also shown in Table 16. The test was performed twice with each triplet for each test sample solution except NK-56 and NK-53, and the average was obtained. Further, when only NGF was added to this experimental system (5 ng / ml), the neurite extension rate was about 5%.
  • R 7 to R 9 represent the same or different aliphatic hydrocarbon groups.
  • X 3 - represents an appropriate counter anion
  • m represents an integer of either charge become 1 or 2 to balance the charge of the cation.
  • the above experimental results are represented by the general formula 3 such as NK-19, NK-53, NK-150, etc., and the compounds having 3 to 10 carbon atoms in the side chain alkyl group, especially represented by the general formula 6. Since compounds having 3 to 10 carbon atoms in the side chain alkyl group have physiological functions such as neurotrophic factor-like action and neurodegeneration inhibitory action, these compounds can be used for Alzheimer's disease, cerebellar ataxia, etc. It is useful as an anti-neurodegenerative disease agent.
  • NK-4 analog Neurotrophic factor-like activity of NK-4 analog>
  • the side chain alkyl group (R 4 to R 6 ) Seven types of compounds having 2 to 8 carbon atoms and a counter anion of I ⁇ were synthesized, and as in Experiment 3, the strength of cell proliferation promoting action and neurite extension promoting action on PC12-HS cells was increased. Examined.
  • NK-4 seven types of compounds shown in Table 17, NK-234, NK-26, NK-9815, NK-9694, NK-28 and NK-147, each in DMSO at 5 mg / ml It dissolved so that it might become. This solution was diluted with 10% by volume FBS-added D-MEM so that the final concentrations of the compounds were as shown in Table 17 or 18, respectively, to prepare test sample solutions.
  • NK-19 analogs NK-13, NK-392, NK-19 and NK-150 are dissolved in DMSO to a concentration of 5 mg / ml, so that the concentration of the compound becomes the concentration shown in Table 17 or 18.
  • Table 17 shows the results of the cell growth promoting action
  • Table 18 shows the results of the neurite extension promoting action.
  • R 4 to R 6 represent the same or different aliphatic hydrocarbon groups.
  • X 2 ⁇ represents an appropriate counter anion
  • m represents an integer that is either 1 or 2 that has a charge balanced with the charge of the cation moiety.
  • the NK-4 analog represented by the general formula 2 has a compound having 3 to 8 carbon atoms in the side chain alkyl group, and has a cell growth equivalent to or higher than that of NK-4.
  • the cell growth promoting action was particularly strong for compounds having 4 to 6 carbon atoms in the side chain alkyl group.
  • the neurite outgrowth promoting action a strong activity was observed in the compounds having 4 and 5 carbon atoms in the side chain alkyl group.
  • a compound represented by the general formula 3 and having a side chain alkyl group having 2 to 8 carbon atoms particularly, an NK— represented by the general formula 3 and having a side chain alkyl group having 6 to 8 carbon atoms. It was revealed that 19 analog compounds showed strong cell proliferation and neurite outgrowth promoting activity.
  • NK-4, NK-9694, NK-19, and NK-150 significantly inhibited cell damage caused by 6-hydroxydopa.
  • 6-Hydroxydopa is a catecholaminergic neuron-selective neurotoxin used as an in vivo and in vitro model of Parkinson's disease, and as a result, NK-4 analogs and NK-19 analogs are therapeutic agents for Parkinson's disease. It suggests that it can be used. Also, under these experimental conditions, commercially available edaravone or donepezil has an inhibitory action at a concentration of 1 ⁇ g / ml or less at which the cytotoxicity caused by 6-hydroxydopa is not inhibited. Therefore, NK-4, NK It is concluded that -9694, NK-19, and NK-150 are more effective in suppressing cell damage caused by 6-hydroxydopa than edaravone or donepezil.
  • amyloid ⁇ fragment disorder and hydrogen peroxide disorder using primary neurons (neurons, astrocytes and microglia cells) prepared from rat fetal cerebral cortex instead of PC-12HS cells.
  • primary neurons neurons, astrocytes and microglia cells
  • NK-4, NK-26, NK-234, NK-19 and NK-150 were examined for their effects on these cells, and these compounds have activity to inhibit amyloid ⁇ fragment damage and hydrogen peroxide damage to primary neurons It has been found.
  • these compounds were found to suppress the production of NO produced by microglia cells in the presence of LPS.
  • NK-4 analog or NK-19 analog Effect of NK-4 analog or NK-19 analog on rat model of cerebral infarction>
  • the effects of NK-4, NK-26, and NK-15, which were confirmed to have cell growth promoting action and neurite promoting action on PC-12HS cells, on cerebral infarction were examined using human cerebral infarction model rats. That is, according to Experiment 7, SD rats (manufactured by Charles River Japan, male, 8 weeks old, body weight 280 to 330 g) were embolized in the middle cerebral artery.
  • NK-150, NK-26, and NK-4 were each dissolved in DMSO at a concentration of 5 mg / ml, filtered through a membrane filter with a pore size of 0.45 ⁇ m, and further 2.5 to 0.05 mg / ml in DMSO. What was adjusted to the concentration was stored in the dark.
  • These compound solutions were diluted 250-fold with saline at the time of use and administered to the embolized rat through the tail vein twice after 1 hour of occlusion and at the time of reopening (fluid amount: 5 ml / kg / body weight).
  • NK-4 was prepared as a 10 mg / ml solution, diluted 250 to 167 times, and administered from the tail vein (fluid volume: 5 ml / kg / body weight).
  • physiological saline was administered as a negative control
  • an existing drug edaravone manufactured by Mitsubishi Tanabe Pharma Corporation, trade name “Radicut”
  • DMSO DMSO
  • a behavioral score was obtained by the same method as in Experiment 7 to evaluate neurological symptoms.
  • the actual volume value was obtained by dividing by the swelling rate.
  • the results and the group composition are shown in Table 20.
  • NK-4 analog As described above, the AchE activity inhibitor is used as a therapeutic agent for Alzheimer-type dementia. Therefore, assuming that the NK-4 analog is applied to Alzheimer's disease, the strength of the Ache activity inhibitory action of the NK-4 analog was compared. That is, using the four NK-4 analogs having 2 to 5 carbon atoms in the side chain alkyl group of the compound represented by the general formula 2 used in Experiment 11, the remaining AchE activity ( %). In addition, the remaining percentage of Ache activity (%) was measured using NK-19 analog NK-150. Table 21 also shows the IC50 values (compound concentrations that inhibit the Ache activity used in the experiment by 50%) calculated based on the results and the residual activity rate.
  • IC50 values compound concentrations that inhibit the Ache activity used in the experiment by 50%
  • NK-4 analogs showed a tendency that the residual ratio of the Ache activity increased as the number of carbon atoms of the side chain alkyl group increased.
  • NK-4 and NK-234 are useful as a therapeutic agent for Alzheimer-type dementia.
  • Ache activity remaining rate (%) of donepezil hydrochloride (trade name “Aricept”) marketed for the treatment of Alzheimer-type dementia as an Ache activity inhibitor was measured in the same experimental system, the IC 50 was 0.9 ⁇ g. / Ml, NK-4 has almost the same activity of inhibiting Ache activity as donepezil hydrochloride.
  • NK-4 analog and NK-19 analog on model mice of human Alzheimer type dementia The above experiments suggested that NK-4 can be used as a therapeutic agent for Alzheimer's dementia.
  • NK-4 analogs and NK-19 analogs were modeled on human Alzheimer's model mice. The effect was verified.
  • NK-4, NK-234, NK-26, NK-19 and NK-150 were used as test samples.
  • physiological saline 200 ⁇ l / animal
  • Donepezil hydrochloride was used as control 2.
  • Each test sample was dissolved in DMSO to a concentration of 5 mg / ml, and diluted with physiological saline for administration.
  • mice manufactured by Charles River Japan, male, 5 weeks old, body weight 25 to 30 g
  • Chloral hydrate SIGMA, 350 mg / kg, body weight, intraperitoneal administration
  • Anesthetized mice were fixed on the back, and a midline incision was made on the head.
  • Evans blue solution (0.3 ⁇ g / 0.3 ⁇ l) is administered in advance for the insertion site instead of the amyloid ⁇ fragment solution, and the lateral ventricle, dorsal third ventricle, ventral third brain of the left and right forehead cross section It was confirmed that coloring was observed in the room. After the administration, the scalp was sutured, and one of the compounds was intraperitoneally administered once a day for 13 days from the next day, and behavioral evaluation was performed by the following method. The results and group composition are shown in Table 22.
  • the novel object recognition test uses the characteristics of the mouse when it likes the novelty, and unlike many other learning evaluation systems, it does not use artificial reinforcement factors.
  • the test consisted of three departments: acclimatization, training trials, and retention trials, which were performed 6-8 days after amyloid ⁇ fragment was administered into the ventricle.
  • An open field experimental device (40 cm long, 30 cm wide, 30 cm high) with wood chips on the floor was installed in a noise-free place under about 1,000 lux lighting. First, on the 6th day, the mouse was placed in the center of the experimental apparatus without the search object and allowed to search freely for 10 minutes (acclimation).
  • the object identification index is the ratio of the time allotted to the search for new objects to the total search time, and if the animal stores the object once searched, the value of the object identification index becomes large, If not stored, the value becomes smaller.
  • ⁇ Passive avoidance test> A step-through type using the property that the mouse prefers the dark room is adopted as an index of memory, which is the avoidance behavior shown to the aversive stimulation (electrical stimulation) once experienced by the animal.
  • the movement time to the dark room side when the mouse was put in the light room side of the device where the light room and dark room were connected by a door was used as an index of memory.
  • the passive avoidance test was performed 9 to 12 days after amyloid ⁇ fragment was administered into the ventricle. On the 9th day, the light room (1,000 lux, length 30 cm, width 30 cm, height 15 cm) was placed in a dark room (length 30 cm, width 30 cm, height 15 cm) for 2 minutes to acclimatize.
  • the acclimatization was similarly performed on the 10th day.
  • the mouse was first placed in the center of the light room, and at the same time the mouse moved into the dark room, the door between the light room and the dark room was closed, and electrical stimulation was applied (0.8 mA, 1 Seconds). After 24 hours (day 12), the mouse was placed in the center of the light room again as in the previous day, and the moving time (seconds) to the dark room was measured as a passive avoidance response. If the aversive stimulus by energization is memorized, the passive avoidance reaction becomes longer.
  • NK-4 is 500 ⁇ g / kg ⁇ body weight
  • NK-234 is 500 ⁇ g / kg ⁇ body weight
  • NK-26 is 50 ⁇ g / kg ⁇ body weight
  • NK-19 is 500 ⁇ g / kg ⁇ body weight
  • NK ⁇ In mice administered with 150 at 500 ⁇ g / kg ⁇ body weight, a significant improvement was observed in the novel object recognition test compared to mice administered with amyloid ⁇ fragment alone.
  • NK-4 is 50 ⁇ g / kg ⁇ body weight
  • NK-4 is 500 ⁇ g / kg ⁇ body weight
  • NK-234 is 500 ⁇ g / kg ⁇ body weight
  • NK-26 is 50 ⁇ g / kg ⁇ body weight
  • NK-26 is 500 ⁇ g / kg ⁇ body weight.
  • Significant improvement was observed in the passive avoidance test in mice administered with body weight and NK-19 at 500 ⁇ g / kg / body weight, compared with mice administered with amyloid ⁇ fragment alone.
  • NK-234, NK-26, NK-19, and NK-150 all have an action to improve cognitive impairment caused by amyloid ⁇ peptide.
  • NK-4 has the strongest cognitive impairment improving effect.
  • mice wild type, female, 10 weeks old, body weight 15 to 23 g
  • physiological saline was intraperitoneally injected 5 times a week.
  • physiological saline 200 ⁇ l / mouse
  • APP Tg mice were administered donepezil hydrochloride 5 times a week for 12 weeks.
  • ⁇ Water maze test method> A circular pool with a diameter of 130 cm was filled with water colored with white ink to a depth of 20 cm, and the water temperature was maintained at 23 ⁇ 1 ° C. with a water tank heater.
  • the pool was divided into four parts, and an evacuation platform was installed at a position 10 cm from the side of the pool so as to be 2 cm below the surface of the water. The platform position was fixed until the end of the test. From the day after the end of the passive avoidance test, the mouse was released on the surface of the pool toward the side of the pool, and the time taken to reach the platform hidden under the surface of the water was measured.
  • the starting position was 10 cm away from the central part of one of the fractions of the pool divided into 4 centimeters, and was randomly changed for each trial.
  • the mouse After searching the platform freely for 2 minutes, if the mouse could not reach the platform within 2 minutes, it was guided to the platform, allowed to stay on the platform for 30 seconds and then transferred to a cage with paper towels.
  • the second test started 1 minute after the end of the first test. This test was conducted for 4 consecutive days, and the average value of the two trials was taken as the value of the day.
  • mice administered NK-4 markedly improved the cognitive impairment of APP Tg mice in any of the object identification index, passive avoidance reaction, and water maze test. Moreover, the improvement effect was significantly stronger than that of commercially available Alzheimer-type dementia therapeutic agent donepezil hydrochloride. This result strongly suggests that NK-4 can be used as a therapeutic agent for Alzheimer-type cognitive impairment. During the study, no side effects believed to be caused by administration of NK-4 were observed.
  • NK-4 Effect of NK-4 on cerebrovascular dementia model mice>
  • the above-mentioned experiment confirmed that NK-4 is effective in improving dyskinesia and Alzheimer-type cognitive impairment due to cerebral infarction. It was. Specifically, 31 C57BL / 6J mice (manufactured by Claire Japan, male, 12 weeks old) were preliminarily raised for 1 week, and then atropine (0.3 mg / kg, subcutaneous administration) was pre-administered to 21 mice, followed by pentobarbital sodium. (50 mg / kg) was intraperitoneally administered and anesthetized, and a permanent ligation operation was performed on the right common carotid artery (refer to Japanese Patent Application Laid-Open No.
  • mice After the operation, all mice were reared alone, and were bred with free eating and drinking water. Of the 21 animals subjected to ligation surgery, 10 animals were reared as they were (ligation group), and the remaining 11 animals were administered NK-4 (administration group). In addition, 10 animals that had not undergone surgery were used as controls (no surgery group). From the second day after the operation, physiological saline was administered intraperitoneally to the non-operative group and ligation group, and NK-4 (100 ⁇ g / kg) was intraperitoneally administered daily (5 days a week) to the NK-4 administration group. A novel object recognition test was performed in the same manner as in Experiment 15 on the third and fourth weeks of the operation. The results are shown in Table 24.
  • NK-4 can be used as a therapeutic agent for vascular cognitive impairment.
  • no side effects believed to be caused by administration of NK-4 were observed.
  • the above experimental results are represented by the general formula 3 such as NK-19, NK-53, NK-150, etc., and are represented by the general formula 2 like the compounds having 3 to 10 carbon atoms in the side chain alkyl group. Since compounds having 2 to 8 carbon atoms in the side chain alkyl group have an inhibitory effect on neurodegeneration, all of these compounds have cognitive impairment due to cerebral infarction, motor impairment, Alzheimer type cognitive impairment, blood vessel It is useful as an anti-neurodegenerative disease agent for the treatment of sexual cognitive impairment and cerebellar ataxia.
  • NK-4, NK-234, and NK-26 which are compounds represented by the general formula 2 and have 2 to 4 carbon atoms in the side chain alkyl group, have a strong neurodegeneration inhibitory effect, and particularly NK-4. It is concluded that it is excellent in improving various symptoms associated with neurodegeneration such as cognitive impairment due to cerebral infarction, motor impairment, Alzheimer type cognitive impairment, vascular cognitive impairment, and Parkinson's disease.
  • anti-neurodegenerative disease agent of the present invention will be described with reference to examples, but the present invention is not limited to these examples.
  • ⁇ Liquid preparation for injection> A solution prepared by dissolving 60 g of purified maltose for injection (manufactured by Hayashibara Co., Ltd.) in 370 g of purified water for injection, NK-4 (compound represented by Chemical Formula 2), NK-26 as an active ingredient in 170 g of purified water for injection (Compound represented by chemical formula 1), NK-28 (compound in which the carbon number of the alkyl group (R) in the side chain of the compound represented by general formula 2 is 7), NK-147 (expressed by general formula 2) A compound in which the alkyl group (R) in the side chain of the compound has 8 carbon atoms), NK-19 (a compound represented by Chemical Formula 4), NK-53 (a compound represented by Chemical Formula 5), NK- 150 (compound represented by chemical formula 3), NK-393 (compound in which the side chain alkyl group (R) of the compound represented by general formula 3 has 8 carbon atoms), NK-100 (compound represented by chemical formula
  • All of this product is pyrogen-free and can be used as an anti-neurodegenerative disease agent.
  • the product can be used as a neurodegeneration inhibitor, a nerve cell protective agent, a neurite promoter, or a therapeutic agent for a disease state or neurological dysfunction associated with neurodegeneration.
  • this product is a brain protective agent, brain oxidative disorder inhibitor, ischemic brain disorder inhibitor, cerebral infarction growth inhibitor, brain edema inhibitor, delayed neuronal death inhibitor, brain function normalizing agent, Oxidative stress inhibitor, anti-ulcer agent, blood sugar elevation inhibitor, prevention / treatment agent for ocular diseases, transplanted organ preservative, transplanted tissue / organ necrosis inhibitor, tissue / organ injury preventive / therapeutic agent, radiation damage Prophylactic / therapeutic agent, antitumor agent, tumor metastasis inhibitor, cytopathic marker inhibitor, prophylactic / therapeutic agent for inflammatory diseases and associated tissue disorders, sensory cell, sensory nerve or sensory organ disorder inhibitor, drug addiction Preventive / therapeutic agent, calcium / sodium exchange inhibitor, preventive / therapeutic agent for pain and pruritus, protein kinase stimulator, preventive / therapeutic agent for mitochondrial encephalomyopathy, preventive / therapeutic agent for arterial occlusion / stenosis, blood-brain barrier disruption
  • the remaining 10 animals in a group were intraperitoneally administered with a 10% aqueous solution of sterile maltose (pyrogen-free) from 3 weeks to 10 weeks of age, once daily for 56 days, daily at 0.5 ml / mouse. (Control group).
  • the day after the end of the administration period the body weight of each hamster was measured in the same manner as in Experiment 2, and the rotarod test, the slope endurance test, and the number of falls were measured.
  • Table 19 shows the types of compounds that are active ingredients of the preparations administered to each group and the measurement results.
  • the average body weight of a 3-week-old hamster in the control group was 35.4 g
  • the average body weight of 10-week-old was 122.9 g. No significant difference was found in the average body weight from the control group, and Table 25 shows only the results of the rotarod test, the slope endurance test, and the number of falls.
  • mice treated with amyloid ⁇ fragment > 130 ICR mice (available from Charles River Japan Co., Ltd.) were randomly divided into 13 groups, 10 each.
  • the amyloid ⁇ fragment having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing used in Experiment 3 was aged at 37 ° C. for 4 days and administered into the lateral ventricle of 130 mice (9 nmol / 6 ⁇ l / mouse) ( The administration method is described in “Brain Research”, 706, 181-193 (1996)).
  • each of 10 animals in 12 groups contains any of the 12 compounds prepared in Example 1 as active ingredients.
  • any one of the formulations was intraperitoneally administered once a day until the 8th day, daily at 0.3 ml / animal.
  • the remaining 10 animals in 1 group were intraperitoneally administered with a 10% aqueous solution of maltose (pyrogen-free) once a day until the 8th day (control group).
  • a novel object recognition test (see, for example, Japanese Patent Application Laid-Open No. 2008-193941) is performed on the 8th day after administration of amyloid ⁇ fragment.
  • the average of the search time extension ratio is obtained and shown in Table 26 together.
  • mice were dissected and brains were collected, tissue specimens were prepared by a conventional method, and deposition of amyloid ⁇ fragment aggregates was confirmed by Congo red staining or thioflavin T staining, and at the same time, hematoxylin-eosin The degree of degeneration or occlusion of pyramidal cells in the hippocampal region involved in cognitive function was observed in the stained or Nissl stained specimens. Hippocampal pyramidal cell degeneration or loss is scored by assessing it in four stages: mild (1), moderate (2), and severe (3), with the amyloid ⁇ fragment non-administered control state being no (0), Table 20 shows the average of 10 mice in each test group.
  • An experimental apparatus (a glass box having a length of 30 cm, a width of 45 cm, and a height of 30 cm) and two objects stored by the mouse were prepared. The day before the test, the mouse was allowed to acclimatize to the environment by freely searching the experimental apparatus for 10 minutes in the absence of an object. On the test day, the following two trials were performed with a trial interval of 60 minutes. In the first trial, two identical objects were placed at both ends of the experimental apparatus and allowed to freely explore the mouse for 10 minutes. In the second trial, one of the objects used in the first trial was replaced with another kind of object, and the mouse was allowed to freely search for 5 minutes.
  • Object identification index (time spent searching for a new object ⁇ time spent searching for an object once stored) / (time spent searching for a new object + searching for an object stored once) Time)).
  • the identification index is the ratio of the time allotted to the search for new objects to the total search time, and the value increases if the animal stores the object once searched, and does not store it The value becomes smaller.
  • pentamethine cyanine dye compounds (NK-4, NK-26, NK-28, NK-147, NK-19, NK-53, NK-150, NK-393, K -100, K-528, K-557, and NK-1516) (Experimental Groups 13 to 21) were more effective against hippocampal pyramidal cell degeneration and cognitive impairment due to amyloid ⁇ fragment administration A strong improvement effect was observed.
  • ⁇ Liquid preparation for injection> A solution prepared by dissolving 60 g of purified maltose for injection (manufactured by Hayashibara Co., Ltd.) in 370 g of purified water for injection, and 2 g of lecithin and an active ingredient NK-4 (compound represented by Chemical Formula 2) in 170 g of purified water for injection, NK-234 (compound in which the alkyl group (R) in the side chain of the compound represented by general formula 2 has 3 carbon atoms), NK-26 (compound represented by chemical formula 1), NK-28 (general formula 2), NK-147 (the number of carbon atoms in the side chain alkyl group (R) of the compound represented by the general formula 2 is 7).
  • NK-19 compound represented by chemical formula 4
  • NK-53 compound represented by chemical formula 5
  • NK-150 compound represented by chemical formula 3
  • NK-393 General The side chain alkyl group of the compound represented by Formula 3 ( )
  • NK-100 compound represented by chemical formula 6
  • NK-528 compound represented by chemical formula 7
  • NK-557 compound represented by chemical formula 8
  • NK-1516 compound represented by the chemical formula 9) (all manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) were mixed with a solution in which 120 mg each was dissolved, and after filtration sterilization, dissolved oxygen Aseptic nitrogen gas was bubbled until the concentration reached about 0.1 ppm, and 1 ml each was dispensed into a brown ampule, and the ampule was sealed under a nitrogen stream.
  • All of this product is pyrogen-free and can be used as an anti-neurodegenerative disease agent.
  • the product can be used as a neurodegeneration inhibitor, a nerve cell protective agent, a neurite promoter, or a therapeutic agent for a disease state or neurological dysfunction associated with neurodegeneration.
  • this product is a brain protective agent, brain oxidative disorder inhibitor, ischemic brain disorder inhibitor, cerebral infarction growth inhibitor, brain edema inhibitor, delayed neuronal death inhibitor, brain function normalizing agent, Oxidative stress inhibitor, anti-ulcer agent, blood sugar elevation inhibitor, prevention / treatment agent for ocular diseases, transplanted organ preservative, transplanted tissue / organ necrosis inhibitor, tissue / organ injury preventive / therapeutic agent, radiation damage Prophylactic / therapeutic agent, antitumor agent, tumor metastasis inhibitor, cytopathic marker inhibitor, prophylactic / therapeutic agent for inflammatory diseases and associated tissue disorders, sensory cell, sensory nerve or sensory organ disorder inhibitor, drug addiction Preventive / therapeutic agent, calcium / sodium exchange inhibitor, preventive / therapeutic agent for pain and pruritus, protein kinase stimulator, preventive / therapeutic agent for mitochondrial encephalomyopathy, preventive / therapeutic agent for arterial occlusion / stenosis, blood-brain barrier disruption
  • Each of the 13 types of anti-neurodegenerative disease agents prepared in Example 2 was administered as a single 0.5 ml / mouse intraperitoneally to 10 ddy mice (average body weight 25.6 g) for 1 week after administration.
  • 10 dmal mice (average body weight 26.3 g) were administered with 10% maltose solution containing 0.2% lecithin intraperitoneally as a control.
  • the LD 50 of the compound formulated as an active ingredient of the 13 types of anti-neurodegenerative disease agents prepared in Example 2 is 3.9 mg / kg ⁇ body weight or more. It shows that it is safe to administer.
  • ⁇ Powder for injection> A solution prepared by dissolving 60 g of purified maltose for injection (manufactured by Hayashibara Co., Ltd.) in 370 g of purified water for injection, 170 g of purified water for injection, 3 g of polysorbate 80 (sold by NOF Corporation), Compound represented by chemical formula 2), NK-234 (compound in which the carbon number of the alkyl group (R) in the side chain of the compound represented by general formula 2 is 3), NK-26 (represented by chemical formula 1) Compound), NK-28 (a compound in which the alkyl group (R) in the side chain of the compound represented by Formula 2 has 7 carbon atoms), NK-147 (a compound in the side chain of the compound represented by Formula 2) A compound in which the alkyl group (R) has 8 carbon atoms), NK-19 (a compound represented by Formula 4), NK-53 (a compound represented by Formula 5), NK-150 (a compound represented by Formula 3) Compound), NK-393
  • All of these products are pyrogen-free. At the time of use, 2 to 10 ml of purified water for injection or physiological saline is added to the ampoule and dissolved, and then used by methods such as intravenous infusion, subcutaneous administration, and intraperitoneal administration.
  • This product can be used as an anti-neurodegenerative disease agent.
  • the product can be used as a neurodegeneration inhibitor, a nerve cell protective agent, a neurite promoter, or a therapeutic agent for a disease state or neurological dysfunction associated with neurodegeneration.
  • this product is a brain protective agent, brain oxidative disorder inhibitor, ischemic brain disorder inhibitor, cerebral infarction growth inhibitor, brain edema inhibitor, delayed neuronal death inhibitor, brain function normalizing agent, Oxidative stress inhibitor, anti-ulcer agent, blood sugar elevation inhibitor, prevention / treatment agent for ocular diseases, transplanted organ preservative, transplanted tissue / organ necrosis inhibitor, tissue / organ injury preventive / therapeutic agent, radiation damage Prophylactic / therapeutic agent, antitumor agent, tumor metastasis inhibitor, cytopathic marker inhibitor, prophylactic / therapeutic agent for inflammatory diseases and associated tissue disorders, sensory cell, sensory nerve or sensory organ disorder inhibitor, drug addiction Preventive / therapeutic agent, calcium / sodium exchange inhibitor, preventive / therapeutic agent for pain and pruritus, protein kinase stimulator, preventive / therapeutic agent for mitochondrial encephalomyopathy, preventive / therapeutic agent for arterial occlusion / stenosis, blood-brain barrier disruption
  • the anti-neurodegenerative disease agent of the present invention prevents, treats and / or suppresses progression of Parkinson's disease, Parkinson's syndrome, Alzheimer's disease, dementia, or stroke caused by neurodegeneration of nerve cells, and further suppresses neurodegeneration.
  • various pathological conditions and neurological dysfunctions associated with neurodegenerative diseases for example, tremor, rigidity, ataxia, peristalsis, slow movement, posture reflex disorder, autonomic disorder, lunging phenomenon, gait disorder, depression, memory disorder, It is also useful for improving muscle atrophy, muscle weakness, upper limb dysfunction, articulation disorder, dysphagia, respiratory disorder, numbness and paralysis.
  • the anti-neurodegenerative disease agent of the present invention since the anti-neurodegenerative disease agent of the present invention has no side effects even when administered for a long period of time, it is highly safe and can be used with confidence.
  • the present invention is an invention that exhibits such remarkable effects, and is a truly significant invention that contributes greatly to the world.

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Abstract

Disclosed is a novel anti-neurodegenerative disease agent.  The anti-neurodegenerative disease agent comprises a compound represented by general formula (1) as an active ingredient. In general formula (1), R1 to R3 independently represent a hydrogen atom or a proper substituent; Z1 represents a heterocyclic ring, and Z2 represents a heterocyclic ring which is the same as or different from the heterocyclic ring represented by Z1, or an aromatic ring, wherein each of the heterocyclic ring and the aromatic ring may have a substituent; o represents an integer of 0, 1 or 2, and p represents an integer of 0 or 1, wherein p is 1 when o is 0 or 2, p is 0 when o is 1, R1 and R2 do not exist when o is 0, and R3 does not exist and a carbon atom to which R2 is bound and Z2 are bound to each other through a single bond when p is 0; X1 - represents a proper counter anion; and q represents an integer of 1 or 2.

Description

抗神経変性疾患剤Anti-neurodegenerative disease agent
　本発明は、一般式１で表される化合物を有効成分とする抗神経変性疾患剤に関するものである。 The present invention relates to an anti-neurodegenerative disease agent comprising a compound represented by the general formula 1 as an active ingredient.
Figure JPOXMLDOC01-appb-C000008
Figure JPOXMLDOC01-appb-C000008
　一般式１におけるＲ１乃至Ｒ３は、それぞれ独立に、水素原子又は適宜の置換基を表し、Ｚ１は複素環を、また、Ｚ２はＺ１と同じか異なる複素環又は芳香環を表し、それらの複素環及び芳香環は置換基を有していてもよい。ｏは０又は１、２のいずれかである整数を表し、ｐは、０又は１のいずれかである整数を表し、ｏが０又は２のとき、ｐは１であり、ｏが１のとき、ｐは０である。ｏが０の場合、Ｒ１、Ｒ２は存在せず、ｐが０の場合、Ｒ３は存在せず、Ｒ２が結合する炭素とＺ２とは一重結合となる。Ｘｌ －は適宜の対アニオンを表し、ｑは１又は２のいずれかである整数を表す。 R 1 to R 3 in General Formula 1 each independently represent a hydrogen atom or an appropriate substituent, Z 1 represents a heterocyclic ring, and Z 2 represents a heterocyclic ring or an aromatic ring that is the same as or different from Z 1. These heterocyclic rings and aromatic rings may have a substituent. o represents an integer that is either 0, 1, or 2, p represents an integer that is either 0 or 1, p is 1 when o is 0 or 2, and o is 1 , P is 0. When o is 0, R 1 and R 2 do not exist, and when p is 0, R 3 does not exist, and the carbon to which R 2 is bonded and Z 2 form a single bond. X l - represents a suitable counter anion, q is an integer of either 1 or 2.
　神経変性疾患は系統的な神経細胞の変性、脱落に基づく神経回路網の破綻により引き起こされる病態であり、数多くの難病、例えば、アルツハイマー病、パーキンソン病、パーキンソン症候群、脳血管性認知症、前頭側頭葉型認知症、筋萎縮性側索硬化症、進行性核上性麻痺、ハンチントン病、脊髄小脳変性症などが知られている。 Neurodegenerative diseases are pathological conditions caused by systematic neuronal degeneration and disruption of the neural network based on loss, and many intractable diseases such as Alzheimer's disease, Parkinson's disease, Parkinson's syndrome, cerebrovascular dementia, frontal side Known as lobar dementia, amyotrophic lateral sclerosis, progressive supranuclear palsy, Huntington's disease, spinocerebellar degeneration, and the like.
　神経変性疾患の原因である神経変性死のメカニズムには多くの分子群が複雑に関与し、それらの発現、機能異常が生じているものと予想される。しかしながら、その分子病態については解明されたものはほとんどなく、有効な神経変性の抑制方法も確立されていない。病因を取り除く治療に加えて重要なのが、神経回路網を再構築させることである。例えば、アミロイドβペプチドの細胞毒性がその一因と考えられているアルツハイマー病では、神経突起（軸索及び樹状突起）の萎縮とシナプスの減少が、神経機能が損なわれる引き金であり、逆に、その引き金が引かれた後でも、変性しきっていない神経細胞や変性を免れて生き残っている神経細胞を活性化して、神経突起を伸展させ、シナプスを回復できれば、神経機能を回復できるといわれている。しかしながら、損傷をうけた末梢神経系の軸索は再生するものの、中枢神経系では軸索は末梢神経を移植するなどの処置をしないと再生は起こらないと言われている。 It is expected that many molecular groups are involved in the mechanism of neurodegenerative death, which is the cause of neurodegenerative diseases, and that their expression and dysfunction are occurring. However, little has been elucidated about the molecular pathology, and no effective method for suppressing neurodegeneration has been established. In addition to the treatment to remove the etiology, it is important to rebuild the neural network. For example, in Alzheimer's disease, which is thought to be due to the cytotoxicity of amyloid β peptide, atrophy of neurites (axons and dendrites) and a decrease in synapses are triggers that impair neuronal function. Even after the trigger is triggered, it is said that if the nerve cells that have not degenerated and the nerve cells that survive the degeneration are activated, the neurites are extended, and the synapse can be restored, so that the nerve function can be restored. Yes. However, although damaged axons in the peripheral nervous system regenerate, it is said that regeneration does not occur in the central nervous system unless a treatment such as transplantation of peripheral nerves is performed.
　神経成長因子（ｎｅｒｖｅ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ、以下、「ＮＧＦ」と略記する場合がある。）などの神経栄養因子群に属する蛋白は、神経細胞の分化や生存、シナプスの制御などに関与することが知られているものの、高分子ゆえに血液脳関門を通過し難いなどの点からも、皮下や血管内などの全身投与では、中枢神経の変性に起因する神経変性疾患に対する治療効果があまり期待できない。効果を期待して脳内へ投与するためには、外科的処置が必要となり、患者には大きな肉体的・精神的負担が伴う。 Proteins belonging to the group of neurotrophic factors such as nerve growth factor (hereinafter sometimes abbreviated as “NGF”) are known to be involved in the differentiation and survival of neurons and the control of synapses. However, systemic administration such as subcutaneously or intravascularly cannot be expected to have a therapeutic effect on neurodegenerative diseases caused by central nerve degeneration because it is difficult to cross the blood-brain barrier due to the polymer. In order to administer it into the brain with the expectation of an effect, a surgical procedure is required, and the patient is accompanied by a great physical and mental burden.
　神経変性疾患の臨床症状とは、それぞれの病気により異なるが、軽微なものから重篤なものまで様々であり、代表的なものとしては、例えば、振戦、固縮、無動、寡動、動作緩慢、姿勢反射障害、自律神経障害、突進現象、歩行障害、うつ、記憶障害、筋萎縮、筋力低下、上肢機能障害、構音障害、嚥下障害、呼吸障害、しびれ又は麻痺などが挙げられ、これらはいずれも日常生活を営む上で大きな障害となっている。 Clinical symptoms of neurodegenerative diseases vary depending on each disease, but they vary from minor to severe. Typical examples include tremor, rigidity, agitation, peristalsis, and movement. Slowness, postural reflex disorder, autonomic disorder, lunging phenomenon, gait disorder, depression, memory disorder, muscle atrophy, muscle weakness, upper limb dysfunction, articulation disorder, dysphagia, respiratory disorder, numbness or paralysis, etc. Both are major obstacles in daily life.
　アルツハイマー病やパーキンソン病などに代表される神経変性疾患は、神経細胞に変性を来たす重大な疾患であり、これらの疾患やそれに伴う病態や神経機能障害を改善するために、種々の化合物を有効成分とする治療剤が提案されており（例えば、国際公開ＷＯ９７／０３０７０３号パンフレット、特開平１１－２２８４１７号公報、特開２００６－１４３７０８号公報、特開２００６－３２１７３７号公報を参照）、神経突起伸展促進剤なども提案されている（例えば、特開２００２－２３４８４１号公報を参照）ものの、効果的な疾患の治療法はまだ見出されていない。また、市販されている神経変性疾患の治療剤についても、長期連用には副作用などの点で問題がある場合もある。 Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are serious diseases that cause degeneration of nerve cells, and various compounds are used as active ingredients to improve these diseases and their associated pathological conditions and neurological dysfunction. (See, for example, International Publication No. WO97 / 030703, JP-A-11-228417, JP-A-2006-143708, and JP-A-2006-321737). Although an accelerator or the like has also been proposed (see, for example, Japanese Patent Application Laid-Open No. 2002-234841), an effective disease treatment method has not yet been found. Also, commercially available therapeutic agents for neurodegenerative diseases may have problems in terms of side effects and the like for long-term continuous use.
　医療現場では、患者にとって肉体的・精神的負担の少ない、皮下や血管内などの全身投与により、中枢神経系の神経細胞に作用し、神経細胞を活性化し、神経突起の萎縮を抑制、乃至、神経突起の伸展を促進させて神経変性を抑制し、それに伴う病態や臨床症状を治療することができる新規抗神経変性疾患剤の開発が切望されている。 In the medical field, systemic administration such as subcutaneous or intravascular, which has less physical and mental burden for patients, acts on nerve cells of the central nervous system, activates nerve cells, suppresses neurite atrophy, or Development of a novel anti-neurodegenerative disease agent capable of suppressing neurite outgrowth to suppress neurodegeneration and treating the pathological conditions and clinical symptoms associated therewith is eagerly desired.
　本発明は、新規な抗神経変性疾患剤を提供することを課題とする。 An object of the present invention is to provide a novel anti-neurodegenerative disease agent.
　本発明者等は、上記課題を解決するために、鋭意研究して検索した結果、下記一般式１で表される化合物が優れた神経細胞活性化作用を有し、神経突起伸展促進作用を有することを見出した。さらに、これらの化合物が、細胞傷害因子による神経細胞死の抑制作用を有し、全身投与でも中枢神経系の神経細胞を活性化して神経変性を抑制すると共に、神経変性に起因する症状や病態の発症を遅延乃至改善することを見出して、本発明を完成した。すなわち、本発明は、下記一般式１で表される化合物を有効成分とする抗神経変性疾患剤を主な構成とする。 In order to solve the above-mentioned problems, the present inventors have conducted extensive research and search. As a result, the compound represented by the following general formula 1 has an excellent nerve cell activation action and a neurite extension promoting action. I found out. Furthermore, these compounds have an inhibitory effect on neuronal cell death caused by cytotoxic factors, and even when administered systemically, they activate neurons in the central nervous system to suppress neurodegeneration, as well as symptoms and pathologies caused by neurodegeneration. The present invention was completed by finding that the onset was delayed or improved. That is, the present invention mainly comprises an anti-neurodegenerative disease agent containing a compound represented by the following general formula 1 as an active ingredient.
Figure JPOXMLDOC01-appb-C000009
Figure JPOXMLDOC01-appb-C000009
　一般式１におけるＲ１乃至Ｒ３は、それぞれ独立に、水素原子又は適宜の置換基を表し、Ｚ１は複素環を、また、Ｚ２はＺ１と同じか異なる複素環又は芳香環を表し、それらの複素環及び芳香環は置換基を有していてもよい。ｏは０又は１、２のいずれかである整数を表し、ｐは、０又は１のいずれかである整数を表し、ｏが０又は２のとき、ｐは１であり、ｏが１のとき、ｐは０である。ｏが０の場合、Ｒ１、Ｒ２は存在せず、ｐが０の場合、Ｒ３は存在せず、Ｒ２が結合する炭素とＺ２とは一重結合となる。Ｘｌ －は適宜の対アニオンを表し、ｑは１又は２のいずれかである整数を表す。 R 1 to R 3 in General Formula 1 each independently represent a hydrogen atom or an appropriate substituent, Z 1 represents a heterocyclic ring, and Z 2 represents a heterocyclic ring or an aromatic ring that is the same as or different from Z 1. These heterocyclic rings and aromatic rings may have a substituent. o represents an integer that is either 0, 1, or 2, p represents an integer that is either 0 or 1, p is 1 when o is 0 or 2, and o is 1 , P is 0. When o is 0, R 1 and R 2 do not exist, and when p is 0, R 3 does not exist, and the carbon to which R 2 is bonded and Z 2 form a single bond. X l - represents a suitable counter anion, q is an integer of either 1 or 2.
　本発明の抗神経変性疾患剤は、非経口的に投与することにより、神経細胞の増殖や神経突起の伸展を促進すると共に、栄養や酸素の飢餓やアミロイドβペプチドなどの細胞傷害因子から細胞を保護し、これらの傷害因子により引き起こされる神経変性を抑制して、神経変性疾患に伴う種々の症状（例えば、振戦、固縮、無動、寡動、動作緩慢、姿勢反射障害、自律神経障害、突進現象、歩行障害、うつ、記憶障害、筋萎縮、筋力低下、上・下肢機能障害、構音障害、嚥下障害、呼吸障害、しびれ及び麻痺など）を改善することができる。しかも、有効成分である一般式１で表される化合物の安全性は極めて高い。 The anti-neurodegenerative disease agent of the present invention promotes nerve cell proliferation and neurite outgrowth by parenteral administration, and at the same time removes cells from cytotoxic factors such as nutrient and oxygen starvation and amyloid β peptide. Protects and suppresses neurodegeneration caused by these damaging factors, such as various symptoms associated with neurodegenerative diseases (e.g. tremor, rigidity, agitation, peristalsis, slow movement, posture reflex disorder, autonomic neuropathy, Rush phenomenon, gait disorder, depression, memory disorder, muscle atrophy, muscle weakness, upper / lower limb dysfunction, articulation disorder, dysphagia, respiratory disorder, numbness and paralysis. And the safety | security of the compound represented by General formula 1 which is an active ingredient is very high.
　本発明でいう神経突起とは、神経の細胞体から伸びる軸索及び樹状突起をいう。また、神経突起伸展促進作用とは、神経細胞を活性化して軸索及び／又は樹状突起を伸展させる作用をいい、神経突起の萎縮や減少の抑制作用、神経細胞間のシナプス形成の促進作用やシナプスの減少を抑制する作用を含む。 In the present invention, the neurite means an axon and dendrite extending from a neuronal cell body. The neurite outgrowth promoting action refers to the action of activating nerve cells to extend axons and / or dendrites, the action of suppressing neurite atrophy and decrease, and the action of promoting synapse formation between nerve cells. And the action of suppressing the decrease in synapses.
　本発明でいう神経変性とは、神経細胞、とりわけ、中枢神経系の神経細胞の機能低下、死滅、や減少（脱落）をいい、神経突起の萎縮や減少、シナプスの減少、グリア細胞の機能低下、死滅や減少、網膜細胞の死滅や変性を含む。 Neurodegeneration as used in the present invention refers to a decrease in function, death, or decrease (dropout) of nerve cells, particularly neurons in the central nervous system. Atrophy and decrease of neurites, decrease of synapses, decrease of function of glial cells , Including death and reduction, death and degeneration of retinal cells.
　本発明の抗神経変性疾患剤は、下記一般式１で表される化合物を有効成分とする。 The anti-neurodegenerative disease agent of the present invention contains a compound represented by the following general formula 1 as an active ingredient.
Figure JPOXMLDOC01-appb-C000010
Figure JPOXMLDOC01-appb-C000010
　一般式１におけるＲ１乃至Ｒ３は、それぞれ独立に、水素原子又は適宜の置換基を表し、Ｚ１は複素環を、また、Ｚ２はＺ１と同じか異なる複素環又は芳香環を表し、それらの複素環及び芳香環は置換基を有していてもよい。ｏは０又は１、２のいずれかである整数を表し、ｐは、０又は１のいずれかである整数を表し、ｏが０又は２のとき、ｐは１であり、ｏが１のとき、ｐは０である。ｏが０の場合、Ｒ１、Ｒ２は存在せず、ｐが０の場合、Ｒ３は存在せず、Ｒ２が結合する炭素とＺ２とは一重結合となる。Ｘｌ －は適宜の対アニオンを表し、ｑは１又は２のいずれかである整数を表す。 R 1 to R 3 in General Formula 1 each independently represent a hydrogen atom or an appropriate substituent, Z 1 represents a heterocyclic ring, and Z 2 represents a heterocyclic ring or an aromatic ring that is the same as or different from Z 1. These heterocyclic rings and aromatic rings may have a substituent. o represents an integer that is either 0, 1, or 2, p represents an integer that is either 0 or 1, p is 1 when o is 0 or 2, and o is 1 , P is 0. When o is 0, R 1 and R 2 do not exist, and when p is 0, R 3 does not exist, and the carbon to which R 2 is bonded and Z 2 form a single bond. X l - represents a suitable counter anion, q is an integer of either 1 or 2.
　一般式１におけるＸｌ －は適宜の対アニオンを表し、通常、例えば、弗素イオン、塩素イオン、臭素イオン、沃素イオン、過塩素酸イオン、過沃素酸イオン、六弗化燐酸イオン、六弗化アンチモン酸イオン、六弗化錫酸イオン、燐酸イオン、硼弗化水素イオン、四弗硼素酸イオンなどの無機酸アニオンや、チオシアン酸イオン、ベンゼンスルホン酸イオン、ナフタレンスルホン酸イオン、ナフタレンジスルホン酸イオン、ｐ－トルエンスルホン酸イオン、アルキルスルホン酸イオン、ベンゼンカルボン酸イオン、アルキルカルボン酸イオン、トリハロアルキルカルボン酸イオン、アルキル硫酸イオン、トリハロアルキル硫酸イオン、ニコチン酸イオン、アスパラギン酸イオンなどの有機酸アニオンから選択される。 X l in the general formula 1 - represents an appropriate counter anion, usually, for example, fluorine ion, chlorine ion, bromine ion, iodine ion, perchlorate ion, periodic acid ion, hexafluorophosphate ion, Rokudoruka Inorganic acid anions such as antimonate ions, hexafluorostannate ions, phosphate ions, borofluoride ions, tetrafluoroborate ions, thiocyanate ions, benzenesulfonate ions, naphthalenesulfonate ions, naphthalene disulfonate ions Organic acid anions such as p-toluenesulfonate ion, alkylsulfonate ion, benzenecarboxylate ion, alkylcarboxylate ion, trihaloalkylcarboxylate ion, alkylsulfate ion, trihaloalkylsulfate ion, nicotinate ion, aspartate ion Selected from.
　一般式１で表される化合物としては、より具体的には、一般式２乃至４のいずれかで表されるペンタメチン系シアニン色素及び一般式５で表されるジメチン系スチリル色素などの色素化合物（以下、単に「化合物」という場合がある。）を例示することができる。 More specifically, examples of the compound represented by the general formula 1 include dye compounds such as a pentamethine cyanine dye represented by any one of the general formulas 2 to 4 and a dimethine styryl dye represented by the general formula 5 ( Hereinafter, it may be simply referred to as “compound”).
Figure JPOXMLDOC01-appb-C000011
 一般式２において、Ｒ４乃至Ｒ６は互いに同じか異なる脂肪族炭化水素基を表す。Ｘ２ －は適宜の対アニオンを表し、ｍはカチオン部の電荷とバランスする電荷となる１又は２のいずれかである整数を表す。
Figure JPOXMLDOC01-appb-C000011
 In General Formula 2, R 4 to R 6 represent the same or different aliphatic hydrocarbon groups. X 2 − represents an appropriate counter anion, and m represents an integer that is either 1 or 2 that has a charge balanced with the charge of the cation moiety.
Figure JPOXMLDOC01-appb-C000012
 　一般式３において、Ｒ７乃至Ｒ９は互いに同じか異なる脂肪族炭化水素基を表す。Ｘ３ －は適宜の対アニオンを表し、ｍはカチオン部の電荷とバランスする電荷となる１又は２のいずれかである整数を表す。
Figure JPOXMLDOC01-appb-C000012
 In the general formula 3, R 7 to R 9 represent the same or different aliphatic hydrocarbon groups. X 3 - represents an appropriate counter anion, m represents an integer of either charge become 1 or 2 to balance the charge of the cation.
Figure JPOXMLDOC01-appb-C000013
 　一般式４において、Ｒ１０乃至Ｒ１２は互いに同じか異なる脂肪族炭化水素基を表す。Ｘ４ －は適宜の対アニオンを表し、ｍはカチオン部の電荷とバランスする電荷となる１又は２のいずれかである整数を表す。
Figure JPOXMLDOC01-appb-C000013
 In general formula 4, R 10 to R 12 represent the same or different aliphatic hydrocarbon groups. X 4 − represents an appropriate counter anion, and m represents an integer that is either 1 or 2 that has a charge balanced with the charge of the cation moiety.
Figure JPOXMLDOC01-appb-C000014
 　一般式５において、Ｚ３は複素芳香環を表し、その複素芳香環は置換基を有していてもよい。Ｚ４は芳香環又は複素芳香環を表し、それらの複素芳香環及び芳香環は置換基を有していてもよい。Ｒ１３は脂肪族炭化水素基を表し、その脂肪族炭化水素基は置換基を有していてもよい。Ｒ１４は水素原子又は適宜の置換基を、また、Ｘ５ －は適宜の対アニオンを表す。
Figure JPOXMLDOC01-appb-C000014
 In General Formula 5, Z 3 represents a heteroaromatic ring, and the heteroaromatic ring may have a substituent. Z 4 represents an aromatic ring or a heteroaromatic ring, and the heteroaromatic ring and the aromatic ring may have a substituent. R 13 represents an aliphatic hydrocarbon group, and the aliphatic hydrocarbon group may have a substituent. The R 14 is hydrogen atom or an appropriate substituent and, X 5 - represents a suitable counter anion.
　一般式２乃至５におけるＲ４乃至Ｒ１３で表される脂肪族炭化水素基としては、通常、炭素数が１乃至１２であるものが選択され、２乃至１０のものが好ましく、２乃至９のものがより好ましい。なかでも、一般式２で表される化合物のＲ４乃至Ｒ６の脂肪族炭化水素基の炭素数が２乃至１２、又は、一般式３で表される化合物のＲ７乃至Ｒ９の脂肪族炭化水素基の炭素数が４乃至１０の化合物は、神経変性抑制作用が強いので、特に望ましい。個々の脂肪族炭化水素基としては、例えば、メチル基、エチル基、プロピル基、イソプロピル基、ブチル基、イソブチル基、ｓｅｃ－ブチル基、ｔｅｒｔ－ブチル基、ペンチル基、イソペンチル基、ｔｅｒｔ－ペンチル基、１－メチルペンチル基、２－メチルペンチル基、ヘキシル基、イソヘキシル基、５－メチルヘキシル基、ヘプチル基、オクチル基、ノニル基、デシル基、ウンデシル基、ドデシル基などが挙げられる。また、一般式２乃至５においてＸ２ －乃至Ｘ５ －で表される適宜の対アニオンは、通常、例えば、弗素イオン、塩素イオン、臭素イオン、沃素イオン、過塩素酸イオン、過沃素酸イオン、六弗化燐酸イオン、六弗化アンチモン酸イオン、六弗化錫酸イオン、燐酸イオン、硼弗化水素イオン、四弗硼素酸イオンなどの無機酸アニオンや、チアシアン酸イオン、ベンゼンスルホン酸イオン、ナフタレンスルホン酸イオン、ナフタレンジスルホン酸イオン、ｐ－トルエンスルホン酸イオン、アルキルスルホン酸イオン、ベンゼンカルボン酸イオン、アルキルカルボン酸イオン、トリハロアルキルカルボン酸イオン、アルキル硫酸イオン、トリハロアルキル硫酸イオン、ニコチン酸イオン、アスパラギン酸イオンなどの有機酸アニオンから選択される。 As the aliphatic hydrocarbon group represented by R 4 to R 13 in the general formulas 2 to 5, those having 1 to 12 carbon atoms are usually selected, those having 2 to 10 are preferable, and those having 2 to 9 are preferable. Those are more preferred. Among them, the aliphatic hydrocarbon group of R 4 to R 6 of the compound represented by the general formula 2 has 2 to 12 carbon atoms, or the compound of the compound represented by the general formula 3 of R 7 to R 9 is aliphatic. A compound having 4 to 10 carbon atoms in the hydrocarbon group is particularly desirable because it has a strong inhibitory effect on neurodegeneration. Examples of the individual aliphatic hydrocarbon group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, and a tert-pentyl group. 1-methylpentyl group, 2-methylpentyl group, hexyl group, isohexyl group, 5-methylhexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group and the like. In addition, suitable counter anions represented by X 2 − to X 5 − in the general formulas 2 to 5 are usually, for example, fluorine ion, chlorine ion, bromine ion, iodine ion, perchlorate ion, periodate ion , Inorganic acid anions such as hexafluorophosphate ion, hexafluoroantimonate ion, hexafluorostannate ion, phosphate ion, borofluoride ion, tetrafluoroborate ion, thiocyanate ion, benzenesulfonate ion , Naphthalene sulfonate ion, naphthalene disulfonate ion, p-toluene sulfonate ion, alkyl sulfonate ion, benzene carboxylate ion, alkyl carboxylate ion, trihaloalkyl carboxylate ion, alkyl sulfate ion, trihaloalkyl sulfate ion, nicotinic acid Selected from organic acid anions such as ions and aspartate ions The
　一般式２で表される化合物としては、具体的には、例えば、化学式１で表される化合物（以下、「ＮＫ－２６」という場合がある。）、化学式２で表される化合物（以下、「ＮＫ－４」という場合がある。）や一般式２の側鎖のアルキル基（Ｒ４乃至Ｒ６）の炭素数が３の化合物（以下、「ＮＫ－２３４」という場合がある。）を例示することができる。なお、本明細書中のＮＫ番号に対応する化合物の構造は、例えば、『感光色素表』、感光色素研究所発行（１９６９年）や、『ケミカル　アブストラクト　インデックス　ガイド（ＣＨＥＭＩＣＡＬ　ＡＢＳＴＲＡＣＴ　Ｉｎｄｅｘ　Ｇｕｉｄｅ）（Ｎ－Ｚ）』、１５３１Ｇ乃至１５３６Ｇ頁（１９９４年）などにも記載されている。 Specific examples of the compound represented by the general formula 2 include a compound represented by the chemical formula 1 (hereinafter sometimes referred to as “NK-26”) and a compound represented by the chemical formula 2 (hereinafter, referred to as “NK-26”). Or a compound having 3 carbon atoms in the side chain alkyl group (R 4 to R 6 ) of the general formula 2 (hereinafter sometimes referred to as “NK-234”). It can be illustrated. The structure of the compound corresponding to the NK number in this specification is, for example, “Photosensitive Dye Table”, published by Photosensitive Dye Research Institute (1969), or “Chemical Abstract Index Guide” (N- Z) ”, pages 1531G to 1536G (1994).
Figure JPOXMLDOC01-appb-C000015
  
Figure JPOXMLDOC01-appb-C000015
  
Figure JPOXMLDOC01-appb-C000016
Figure JPOXMLDOC01-appb-C000016
　一般式３で表される化合物としては、具体的には、例えば、化学式３で表される化合物（以下、「ＮＫ－１５０」という場合がある。）や化学式４で表される化合物（以下、「ＮＫ－１９」という場合がある。）を例示することができる。さらには、ＮＫ－１９の対アニオン（Ｉ－）をＣｌ－に替えた化学式５で表される化合物（以下、「ＮＫ－５３」という場合がある。）も、ＮＫ－１９と同様に有利に利用できる。 Specific examples of the compound represented by the general formula 3 include, for example, a compound represented by the chemical formula 3 (hereinafter sometimes referred to as “NK-150”) and a compound represented by the chemical formula 4 (hereinafter, referred to as “NK-150”). "NK-19" may be mentioned). Furthermore, the counter anion of the NK-19 (I -) of Cl - in the compound represented by Chemical Formula 5 for changing (. Which hereinafter may be referred to as "NK-53"), similar to the NK-19 advantageously Available.
Figure JPOXMLDOC01-appb-C000017
Figure JPOXMLDOC01-appb-C000017
Figure JPOXMLDOC01-appb-C000018
Figure JPOXMLDOC01-appb-C000018
Figure JPOXMLDOC01-appb-C000019
Figure JPOXMLDOC01-appb-C000019
　一般式４で表される化合物としては、具体的には、例えば、化学式６で表される化合物（以下、「ＮＫ－１００」という場合がある。）を例示することができる。 Specific examples of the compound represented by the general formula 4 include a compound represented by the chemical formula 6 (hereinafter sometimes referred to as “NK-100”).
Figure JPOXMLDOC01-appb-C000020
Figure JPOXMLDOC01-appb-C000020
　一般式５で表される化合物としては、化学式７乃至９のいずれかで表される化合物（以下、それぞれ、「ＮＫ－５２８」、「ＮＫ－５５７」、「ＮＫ－１５１６」という場合がある。）を例示することができる。
Figure JPOXMLDOC01-appb-C000021
 The compound represented by the general formula 5 may be a compound represented by any one of the chemical formulas 7 to 9 (hereinafter referred to as “NK-528”, “NK-557”, and “NK-1516”, respectively). ).
Figure JPOXMLDOC01-appb-C000021
Figure JPOXMLDOC01-appb-C000022
Figure JPOXMLDOC01-appb-C000022
Figure JPOXMLDOC01-appb-C000023
Figure JPOXMLDOC01-appb-C000023
　化学式１乃至９のいずれかで表される化合物は、いずれも、神経細胞活性化作用、神経突起伸展促進作用に加えて、飢餓、ラジカル、アミロイドβペプチドなどの細胞傷害因子から神経細胞を保護して細胞死や神経突起の萎縮を抑制する作用も有しているので、本発明の抗神経変性疾患剤の有効成分としてより望ましく、その作用効果の強さの点からは、ＮＫ－２６（化学式１で表される化合物）、ＮＫ－４（化学式２で表される化合物）、ＮＫ－２３４（一般式２の側鎖のアルキル基の炭素数が３の化合物）、ＮＫ－１５０（化学式３で表される化合物）が特に望ましい。アセチルコリンエステラーゼ（ＡｃｈＥ）活性抑制作用の強さ、脳内への移行性、製剤化の容易性などを加味すると、ＮＫ－４やＮＫ－２３４が望ましく、ＮＫ－４が特に望ましい。 The compounds represented by any one of Chemical Formulas 1 to 9 protect nerve cells from cytotoxic factors such as starvation, radicals, and amyloid β peptide, in addition to the nerve cell activation action and neurite outgrowth promoting action. Therefore, it is more desirable as an active ingredient of the anti-neurodegenerative disease agent of the present invention because of its action to suppress cell death and neurite atrophy. From the viewpoint of the strength of its action effect, NK-26 (chemical formula 1), NK-4 (compound represented by chemical formula 2), NK-234 (compound with 3 carbon atoms in the side chain alkyl group of general formula 2), NK-150 (chemical formula 3) The compound represented) is particularly desirable. NK-4 and NK-234 are desirable, and NK-4 is particularly desirable, taking into account the strength of acetylcholinesterase (AchE) activity inhibitory activity, migration into the brain, ease of formulation, and the like.
　本発明の抗神経変性疾患剤の有効成分として使用する一般式１で表される化合物は、その由来や製法に制限はない。 The compound represented by the general formula 1 used as an active ingredient of the anti-neurodegenerative disease agent of the present invention is not limited in its origin or production method.
　本発明の抗神経変性疾患剤は、一般式１で表される化合物、望ましくは、一般式２乃至４のいずれかで表されるペンタメチン系シアニン色素及び／又は一般式５で表されるジメチン系スチリル色素を、１種又は２種以上含有してなる。 The anti-neurodegenerative disease agent of the present invention is a compound represented by the general formula 1, preferably a pentamethine cyanine dye represented by any one of the general formulas 2 to 4 and / or a dimethine represented by the general formula 5. It contains one or more styryl dyes.
　本発明の抗神経変性疾患剤は、必要に応じて、有効成分である一般式１で表される化合物に加えて、製剤学的に許容される食品分野、化粧品分野、医薬品分野、医薬部外品分野で使用される成分の１種又は２種以上を配合した製剤の形態で提供される。 The anti-neurodegenerative disease agent of the present invention, if necessary, in addition to the compound represented by the general formula 1 which is an active ingredient, is pharmaceutically acceptable in the food field, cosmetic field, pharmaceutical field, quasi drug It is provided in the form of a preparation containing one or more components used in the product field.
　製剤学的に許容される成分としては、例えば、添加剤、賦形剤、崩壊剤、滑沢剤、安定化剤、界面活性剤、防腐剤（抗菌剤）、香料、増粘剤、抗酸化剤、キレート剤、ビタミン類、アミノ酸類、水性媒体、糖質、水溶性高分子、ｐＨ調整剤、発泡剤、医薬品・医薬部外品・化粧品・食品用の添加剤、医薬用・医薬部外品用の有効成分など例示することができ、これらの成分の１種又は２種以上を適宜組み合わせて配合し、目的とする剤型に応じて、常法により製造すればよい。 Examples of pharmaceutically acceptable ingredients include additives, excipients, disintegrants, lubricants, stabilizers, surfactants, preservatives (antibacterial agents), fragrances, thickeners, and antioxidants. Agents, chelating agents, vitamins, amino acids, aqueous media, sugars, water-soluble polymers, pH adjusters, foaming agents, pharmaceuticals, quasi drugs, cosmetics, food additives, pharmaceuticals, quasi drugs The active ingredient for goods etc. can be illustrated, What is necessary is just to mix | blend combining suitably 1 type (s) or 2 or more types of these components, and to manufacture by a conventional method according to the target dosage form.
　また、本発明の抗神経変性疾患剤は、一般式１で表される化合物以外の神経突起伸展促進剤や、神経変性疾患やそれに起因する病態や神経機能障害の治療剤との併用も有利に実施できる。具体的には、例えば、脳血管障害（例えば、脳卒中、脳梗塞（例えば、脳血栓、脳塞栓など）、一過性脳虚血発作、再灌流障害、脳出血（例えば、高血圧性脳内出血、クモ膜下出血など）など）・脳腫瘍（例えば、星状膠細胞腫、脳膿瘍など）・血液量減少性ショック・外傷性ショック・頭部損傷及び／又は脳脊髄外傷（例えば、脳挫傷・貫入・せん断・圧迫・裂傷、分娩時外傷、乳児むち打ち揺さぶり症候群など）に伴う神経機能障害の治療薬、神経変性疾患（例えば、パーキンソン病、パーキンソン症候群、線条体黒質変性症、ハンチントン病、舞踏病－無定位運動症、進行性核上麻痺、びまん性レビー小体病、大脳皮質基底核変性症、アルツハイマー病、老年性認知症、ピック病、前頭側頭葉型認知症、家族性認知症、脊髄小脳変性症（例えば、オリーブ橋小脳萎縮症、晩発性小脳皮質萎縮症、家族性脊髄小脳失調症（例えば、マッカードジョセフ病など）、歯状核赤核淡蒼球ルイ体萎縮症、家族性痙性対麻痺、フリードライヒ病など）など）の治療薬、運動神経病（例えば、筋萎縮性側索硬化症、家族性筋萎縮性側索硬化症など）の治療薬、脱髄性疾患（例えば、多発性硬化症、汎発硬化症、急性散在性脳脊髄炎、急性小脳炎、横断性脊髄炎、ギラン・バレー症候群など）の治療薬、感染症に伴う脳脊髄疾患（例えば、髄膜炎、インフルエンザ脳症、クロイツフェルト－ヤコブ病、エイズ脳症による認知など）の治療薬、毒物（ヒ素、カドミウム、有機水銀、サリン、ソマン、タブン、ＶＸガスなど）・放射線などによる神経機能障害の治療薬、精神疾患（例えば、神経症、心身症、不安、統合失調症、躁うつ病など）の治療薬、てんかんの治療薬、メージ症候群の治療薬、ジストニアの治療薬、ダウン症の治療薬及び／又は睡眠障害（例えば、過眠、ナルコレプシー、睡眠時無呼吸症候群など）の治療薬、糖尿病の治療薬、糖尿病合併症の治療薬及び／又は高脂血症の治療薬、ドーパミン受容体作動薬（ドーパミン受容体刺激薬）、ドーパミン遊離促進薬（ドーパミン分泌促進薬あるいはドーパミン放出促進薬）、ドーパミン取り込み阻害薬、ドーパミン作用薬、中枢性抗コリン薬、芳香族Ｌ－アミノ酸脱炭酸酵素阻害薬（ＤＣＩ）、モノアミン酸化酵素（ＭＡＯ－Ｂ）阻害薬、カテコール－Ｏ－メチルトランスフェラーゼ（ＣＯＭＴ）阻害薬、ノルエピネフリン（ノルアドレナリン）補充薬、アセチルコリンエステラーゼ阻害薬、ＮＭＤＡ（Ｎ－メチル－Ｄ－アスパラギン酸）受容体拮抗薬、ＡＭＰＡ（２－アミノ－３－（メチル－３－ヒドロキシイソオキサゾール－４－イル）プロパン酸）／カイニン酸受容体拮抗薬、ＧＡＢＡ受容体調節薬、アデノシンＡ２Ａ受容体遮断薬、ニコチン受容体調節薬、神経型一酸化窒素合成酵素（ｎ－ＮＯＳ）阻害薬、βアミロイドタンパクの産生、分泌、蓄積、凝集及び／又は沈着抑制薬（例えば、βセクレターゼ阻害薬、γセクレターゼ阻害作用薬、アミロイドβタンパク凝集抑制薬、アミロイドβタンパク分解酵素、アミロイドβ用ワクチンなど）、アポトーシス阻害薬、神経分化・再生促進薬、神経栄養因子（例えば、ニューロトロフィン、ＴＧＦ－βスーパーファミリー、ニューロカインファミリー、ＮＧＦなどの増殖因子など）、その他の脳機能賦活薬（例えば、脳代謝賦活薬、脳循環改善薬など）、Ｒｈｏ－キナーゼ阻害薬、利尿薬（例えば、チアジド系利尿薬、ループ利尿薬、カリウム保持性利尿薬など）、β受容体遮断薬、カルシウムチャネル遮断薬（カルシウム拮抗薬）、アンジオテンシン変換酵素（ＡＣＥ）阻害薬、アンジオテンシンＩＩ受容体拮抗薬、ナトリウムチャネル遮断薬、カリウムチャネル開口薬、抗血小板薬、抗血液凝固薬、血栓溶解薬、トロンボキサンＡ２合成酵素阻害薬、マトリックスメタロプロテイナーゼ（ＭＭＰ）阻害剤、シクロオキシゲナーゼ（ＣＯＸ）－２阻害薬、非ステロイド性抗炎症薬、ステロイド薬、抗酸化薬、ビタミン類、疾患修飾性抗リウマチ薬、サイトカイン、抗サイトカイン薬（例えば、ＴＮＦ阻害薬など）、ＭＡＰキナーゼ阻害薬、性ホルモン又はその誘導体（例えば、プロゲステロン、エストラジオール、安息香酸エストラジオールなど）、副甲状腺ホルモン（例えば、ＰＴＨなど）及びカルシウム受容体拮抗薬などを例示することができる。これらの薬剤は、本発明の有効成分と混合剤の形態で投与してもよいし、個々の製剤を別々に投与することもできる。 The anti-neurodegenerative disease agent of the present invention is also advantageously used in combination with a neurite outgrowth promoting agent other than the compound represented by the general formula 1, or a therapeutic agent for a neurodegenerative disease, a pathological condition or a neurological disorder resulting therefrom. Can be implemented. Specifically, for example, cerebrovascular disorders (eg, stroke, cerebral infarction (eg, cerebral thrombus, cerebral embolism, etc.), transient ischemic attack, reperfusion injury, cerebral hemorrhage (eg, hypertensive intracerebral hemorrhage, arachnoid membrane) Etc.), brain tumors (eg, astrocytoma, brain abscess, etc.), blood volume reducing shock, traumatic shock, head injury and / or cerebrospinal trauma (eg, brain contusion, penetration, shear)・ Therapeutic agents for neurological dysfunction associated with compression / laceration, labor trauma, infant whiplash, etc., neurodegenerative diseases (eg Parkinson's disease, Parkinson's syndrome, striatal nigra degeneration, Huntington's disease, chorea) Ataxia, progressive supranuclear palsy, diffuse Lewy body disease, basal ganglia degeneration, Alzheimer's disease, senile dementia, Pick's disease, frontotemporal lobe dementia, familial dementia, spinal cord Cerebellar degeneration For example, Olive Bridge cerebellar atrophy, late-onset cerebellar cortical atrophy, familial spinocerebellar ataxia (for example, Maccard Joseph disease, etc.), dentate nucleus red nucleus pallidal Louis atrophy, familial spastic paraplegia ), Therapeutic agents for motor neuropathy (eg, amyotrophic lateral sclerosis, familial amyotrophic lateral sclerosis, etc.), demyelinating diseases (eg, multiple myelopathy) Treatment for sclerosis, general sclerosis, acute disseminated encephalomyelitis, acute cerebellar inflammation, transverse myelitis, Guillain-Barre syndrome, etc., cerebrospinal disease associated with infection (eg meningitis, influenza encephalopathy) , Creutzfeldt-Jakob disease, cognition due to AIDS encephalopathy, etc., toxic drugs (arsenic, cadmium, organic mercury, sarin, soman, soman, tabun, VX gas, etc.), therapeutic agents for neurological dysfunction due to radiation, mental disorders ( For example, neurosis Treatment of psychosomatic disorders, anxiety, schizophrenia, manic depression, etc., treatment of epilepsy, treatment of mage syndrome, treatment of dystonia, treatment of Down syndrome and / or sleep disorders (eg, hypersomnia, narcolepsy) , Sleep apnea syndrome, etc.), diabetes treatment, diabetes complications and / or hyperlipidemia treatment, dopamine receptor agonist (dopamine receptor stimulant), dopamine release promotion Drugs (dopamine secretion promoters or dopamine release promoters), dopamine uptake inhibitors, dopamine agonists, central anticholinergic agents, aromatic L-amino acid decarboxylase inhibitors (DCI), monoamine oxidase (MAO-B) Inhibitor, catechol-O-methyltransferase (COMT) inhibitor, norepinephrine (noradrenaline) supplement, acetylcholine Sterase inhibitor, NMDA (N-methyl-D-aspartic acid) receptor antagonist, AMPA (2-amino-3- (methyl-3-hydroxyisoxazol-4-yl) propanoic acid) / kainic acid receptor antagonist Drugs, GABA receptor modulators, adenosine A2A receptor blockers, nicotine receptor modulators, neuronal nitric oxide synthase (n-NOS) inhibitors, β amyloid protein production, secretion, accumulation, aggregation and / or Anti-deposition agent (eg, β-secretase inhibitor, γ-secretase inhibitor, amyloid β-protein aggregation inhibitor, amyloid β-protease, amyloid β vaccine, etc.), apoptosis inhibitor, neuronal differentiation / regeneration promoter, neurotrophic Factors (eg, neurotrophin, TGF-β superfamily, neurokine family, NGF Which growth factors), other brain function activators (eg cerebral metabolism activators, cerebral circulation improvers, etc.), Rho-kinase inhibitors, diuretics (eg thiazide diuretics, loop diuretics, potassium retention) Diuretics, etc.), beta receptor blockers, calcium channel blockers (calcium antagonists), angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, sodium channel blockers, potassium channel openers, antiplatelet drugs Anticoagulant, thrombolytic, thromboxane A 2 synthase inhibitor, matrix metalloproteinase (MMP) inhibitor, cyclooxygenase (COX) -2 inhibitor, nonsteroidal anti-inflammatory drug, steroid drug, antioxidant , Vitamins, disease-modifying anti-rheumatic drugs, cytokines, anti-cytokine drugs (eg, TNF And MAP kinase inhibitors, sex hormones or derivatives thereof (eg, progesterone, estradiol, estradiol benzoate, etc.), parathyroid hormone (eg, PTH, etc.), calcium receptor antagonists, and the like. . These agents may be administered in the form of a mixture with the active ingredient of the present invention, or individual preparations can be administered separately.
　本発明の抗神経変性疾患剤と併用される神経変性疾患やそれに起因する病態や神経機能障害の治療剤の中で、好ましくは、例えば、脳血管障害（例えば、脳卒中、脳梗塞（例えば、脳血栓、脳塞栓など）、一過性脳虚血発作、脳出血（例えば、高血圧性脳内出血、クモ膜下出血など）など）の治療薬、脳腫瘍の治療薬、脳脊髄外傷（例えば、脳挫傷など）に伴う神経機能障害の治療薬、神経変性疾患（例えば、パーキンソン病、パーキンソン症候群、ハンチントン病、アルツハイマー病、老年性認知症、脊髄小脳変性症など）の治療薬、運動神経病（例えば、筋萎縮性側索硬化症など）の治療薬、脱髄性疾患（例えば、多発性硬化症など）の治療薬、感染症に伴う脳脊髄疾患（例えば、髄膜炎、インフルエンザ脳症、クロイツフェルト－ヤコブ病、エイズ脳症による認知症など）の治療薬、精神疾患（例えば、神経症、心身症、不安、統合失調症、躁うつ病など）の治療薬、てんかんの治療薬、ジストニアの治療薬、糖尿病の治療薬、糖尿病合併症の治療薬及び／又は高脂血症の治療薬、ドーパミン受容体作動薬、ドーパミン遊離促進薬、ドーパミン取り込み阻害薬、ドーパミン作用薬、中枢性抗コリン薬、芳香族Ｌ－アミノ酸脱炭酸酵素阻害薬（ＤＣＩ）、モノアミン酸化酵素（ＭＡＯ－Ｂ）阻害薬、カテコール－Ｏ－メチルトランスフェラーゼ（ＣＯＭＴ）阻害薬、ノルエピネフリン（ノルアドレナリン）補充薬、アセチルコリンエステラーゼ阻害薬、ＮＭＤＡ（Ｎ－メチル－Ｄ－アスパラギン酸）受容体拮抗薬、ＡＭＰＡ（２－アミノ－３－（メチル－３－ヒドロキシイソオキサゾール－４－イル）プロパン酸）／カイニン酸受容体拮抗薬、ＧＡＢＡＡ受容体調節薬（例えば、ＧＡＢＡＡ受容体作動薬など）、ＧＡＢＡＢ受容体調節薬、アデノシンＡ２Ａ受容体遮断薬、βセクレターゼ阻害薬、βアミロイドタンパク凝集抑制薬、アポトーシス阻害薬、神経分化・再生促進薬、神経栄養因子（例えば、ニューロトロフィン、ＴＧＦ－βスーパーファミリー、ニューロカインファミリー、増殖因子など）、その他の脳機能賦活薬（例えば、脳代謝賦活薬、脳循環改善薬など）、Ｒｈｏ－キナーゼ阻害薬、利尿薬（例えば、チアジド系利尿薬、ループ利尿薬、カリウム保持性利尿薬など）、β受容体遮断薬、カルシウムチャネル遮断薬（カルシウム拮抗薬）、アンジオテンシン変換酵素（ＡＣＥ）阻害薬、アンジオテンシンＩＩ受容体拮抗薬、ナトリウムチャネル遮断薬、カリウムチャネル開口薬、抗血小板薬、抗凝固薬、血栓溶解薬、シクロオキシゲナーゼ（ＣＯＸ）－２阻害薬、非ステロイド性抗炎症薬、ステロイド薬、抗酸化薬及びビタミン類が挙げられ、より好ましくは、例えば、脳血管障害（例えば、脳卒中、脳梗塞など）の治療薬、脳脊髄外傷（例えば、脳挫傷など）に伴う神経機能障害の治療薬、神経変性疾患（例えば、パーキンソン病、パーキンソン症候群、ハンチントン病、アルツハイマー病、老年性認知症など）の治療薬、筋萎縮性側索硬化症の治療薬、多発性硬化症の治療薬、精神疾患（例えば、神経症、心身症、不安、統合失調症、躁うつ病など）の治療薬、てんかんの治療薬及び／又はジストニアの治療薬、糖尿病の治療薬、糖尿病合併症の治療薬及び／又は高脂血症の治療薬、ドーパミン受容体作動薬、ドーパミン遊離促進薬、ドーパミン取り込み阻害薬、ドーパミン作用薬、中枢性抗コリン薬、芳香族Ｌ－アミノ酸脱炭酸酵素阻害薬（ＤＣＩ）、モノアミン酸化酵素（ＭＡＯ－Ｂ）阻害薬、カテコール－Ｏ－メチルトランスフェラーゼ（ＣＯＭＴ）阻害薬、ノルエピネフリン（ノルアドレナリン）補充薬、アセチルコリンエステラーゼ阻害薬、ＮＭＤＡ（Ｎ－メチル－Ｄ－アスパラギン酸）受容体拮抗薬、βセクレターゼ阻害薬、βアミロイドタンパク凝集抑制薬、アポトーシス阻害薬、神経分化・再生促進薬、神経栄養因子（例えば、ＮＧＦなどのニューロトロフィン、ＴＧＦ－βスーパーファミリー、ニューロカインファミリー、増殖因子など）、その他の脳機能賦活薬（例えば、脳代謝賦活薬、脳循環改善薬など）、β受容体遮断薬、カルシウムチャネル遮断薬（カルシウム拮抗薬）、アンジオテンシン変換酵素（ＡＣＥ）阻害薬、アンジオテンシンＩＩ受容体拮抗薬、抗血小板薬、抗凝固薬及び血栓溶解薬が挙げられ、とりわけ好ましくは、例えば、脳血管障害（例えば、脳卒中、脳梗塞など）の治療薬、神経変性疾患（例えば、パーキンソン病、パーキンソン症候群、ハンチントン病、アルツハイマー病など）の治療薬、筋萎縮性側索硬化症の治療薬、多発性硬化症の治療薬、てんかんの治療薬及び／又は糖尿病合併症の治療薬、ドーパミン受容体作動薬、ドーパミン遊離促進薬、ドーパミン取り込み阻害薬、ドーパミン作用薬、中枢性抗コリン薬、芳香族Ｌ－アミノ酸脱炭酸酵素阻害薬（ＤＣＩ）、モノアミン酸化酵素（ＭＡＯ－Ｂ）阻害薬、カテコール－Ｏ－メチルトランスフェラーゼ（ＣＯＭＴ）阻害薬、ノルエピネフリン（ノルアドレナリン）補充薬、アセチルコリンエステラーゼ阻害薬、神経栄養因子及びその他の脳機能賦活薬が挙げられる。中でも、ＮＧＦは、本発明で使用する一般式１で表される化合物のもつ神経細胞活性化作用、神経突起伸展促進作用、神経細胞保護作用をはじめとする生理機能が効果的に増強されるので望ましい。 Among the therapeutic agents for neurodegenerative diseases used in combination with the anti-neurodegenerative disease agent of the present invention and the pathological conditions and neurological dysfunctions resulting therefrom, preferably, for example, cerebrovascular disorders (eg, stroke, cerebral infarction (eg, cerebral thrombus) , Cerebral embolism etc.), transient cerebral ischemic attack, cerebral hemorrhage (eg hypertensive intracerebral hemorrhage, subarachnoid hemorrhage etc.), therapeutic agent for brain tumor, cerebrospinal trauma (eg cerebral contusion etc. Drugs for neurological dysfunction associated with, neurodegenerative diseases (eg Parkinson's disease, Parkinson's syndrome, Huntington's disease, Alzheimer's disease, senile dementia, spinocerebellar degeneration), motor neuropathy (eg muscle atrophy) Treatment for demyelinating diseases (eg, multiple sclerosis), cerebrospinal disease associated with infection (eg, meningitis, influenza encephalopathy, Creutzfeldt-Jako) Disease, dementia due to AIDS encephalopathy, etc.), psychiatric disorders (eg, neurosis, psychosomatic disorders, anxiety, schizophrenia, manic depression, etc.), epilepsy, dystonia, diabetes , Diabetic complications and / or hyperlipidemia, dopamine receptor agonist, dopamine release promoter, dopamine uptake inhibitor, dopamine agonist, central anticholinergic, aromatic L Amino acid decarboxylase inhibitor (DCI), monoamine oxidase (MAO-B) inhibitor, catechol-O-methyltransferase (COMT) inhibitor, norepinephrine (noradrenaline) supplement, acetylcholinesterase inhibitor, NMDA (N- Methyl-D-aspartate) antagonist, AMPA (2-amino-3- (methyl-3-hydroxyisothio) Sasol-4-yl) propanoic acid) / kainate receptor antagonists, GABA A receptor modulators (e.g., GABA A receptor agonist), GABA B receptor modulators, adenosine A2A receptor blockers, beta Secretase inhibitor, β-amyloid protein aggregation inhibitor, apoptosis inhibitor, neuronal differentiation / regeneration promoter, neurotrophic factor (eg, neurotrophin, TGF-β superfamily, neurokine family, growth factor), other brains Functional activators (eg, cerebral metabolic activators, cerebral circulation improvers, etc.), Rho-kinase inhibitors, diuretics (eg, thiazide diuretics, loop diuretics, potassium-sparing diuretics), β receptor blockade Drugs, calcium channel blockers (calcium antagonists), angiotensin converting enzyme (ACE) inhibitors, angiotensi II receptor antagonist, sodium channel blocker, potassium channel opener, antiplatelet agent, anticoagulant, thrombolytic agent, cyclooxygenase (COX) -2 inhibitor, nonsteroidal anti-inflammatory agent, steroid drug, antioxidant And more preferably, for example, a therapeutic agent for cerebrovascular disorders (eg, stroke, cerebral infarction, etc.), a therapeutic agent for neurological dysfunction associated with cerebrospinal trauma (eg, cerebral contusion, etc.), neurodegeneration, etc. Therapeutics for diseases (eg Parkinson's disease, Parkinson's syndrome, Huntington's disease, Alzheimer's disease, senile dementia, etc.), amyotrophic lateral sclerosis, multiple sclerosis, mental illness (eg Neurosis, psychosomatic disorders, anxiety, schizophrenia, manic depression, etc.), epilepsy and / or dystonia, diabetes, diabetes Drugs for complications and / or drugs for hyperlipidemia, dopamine receptor agonists, dopamine release promoters, dopamine uptake inhibitors, dopamine agonists, central anticholinergics, aromatic L-amino acid decarboxylase Inhibitor (DCI), monoamine oxidase (MAO-B) inhibitor, catechol-O-methyltransferase (COMT) inhibitor, norepinephrine (noradrenaline) supplement, acetylcholinesterase inhibitor, NMDA (N-methyl-D-asparagine) Acid) receptor antagonist, β-secretase inhibitor, β-amyloid protein aggregation inhibitor, apoptosis inhibitor, neuronal differentiation / regeneration promoter, neurotrophin such as NGF, TGF-β superfamily, neuro Cain family, growth factors, etc.), other brain function activation Drugs (for example, cerebral metabolism activator, cerebral circulation improving drug, etc.), β receptor blocker, calcium channel blocker (calcium antagonist), angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor antagonist, antiplatelet Drugs, anticoagulants and thrombolytic agents, and particularly preferably, for example, therapeutic agents for cerebrovascular disorders (eg, stroke, cerebral infarction, etc.), neurodegenerative diseases (eg, Parkinson's disease, Parkinson's syndrome, Huntington's disease, Alzheimer's disease), amyotrophic lateral sclerosis, multiple sclerosis, epilepsy and / or diabetic complications, dopamine receptor agonist, dopamine release enhancement Drugs, dopamine uptake inhibitors, dopamine agonists, central anticholinergics, aromatic L-amino acid decarboxylase inhibitors (DCI), Noamin oxidase (MAO-B) inhibitor, catechol -O- methyltransferase (COMT) inhibitors, norepinephrine (noradrenaline) replenishers, acetylcholinesterase inhibitors, neurotrophic factors and other brain function activation agents. Among them, NGF effectively enhances the physiological functions of the compound represented by the general formula 1 used in the present invention, such as a nerve cell activation action, a neurite extension promotion action, and a nerve cell protection action. desirable.
　本発明の抗神経変性疾患剤は、通常、非経口用の注射用の製剤などの形態で提供される。有効成分である一般式１で表される化合物は、対象とする注射用の製剤などの非経口投与製剤の組成やその使用目的を勘案して、原料の段階から製品が完成するまでの工程で配合すればよい。その方法としては、例えば、混和、混捏、溶解、融解、分散、懸濁、乳化、逆ミセル化、浸透、晶出、散布、塗布、付着、噴霧、被覆（コーティング）、注入、浸漬、固化、担持などの１種又は２種以上の方法が適宜に選ばれる。 The anti-neurodegenerative disease agent of the present invention is usually provided in the form of a parenteral injection preparation or the like. The compound represented by the general formula 1, which is an active ingredient, is a process from the raw material stage to the completion of the product in consideration of the composition of the parenteral preparation such as an injectable preparation and the purpose of use. What is necessary is just to mix | blend. The methods include, for example, mixing, kneading, dissolving, melting, dispersing, suspending, emulsifying, reverse micellization, infiltration, crystallization, spraying, application, adhesion, spraying, coating (coating), pouring, dipping, solidifying, One or more methods such as loading are appropriately selected.
　注射用製剤などの非経口用の製剤の場合、対象となる疾患や症状などに応じて、通常、パイロジェンを含まない水性媒体に溶解して、皮内、皮下、筋肉内、体腔内（胸腔内、腹腔内など）、血管内又は脳内（脊髄内を含む）へ投与されるので、製剤の形態としては、乾燥製剤であってもよく、液剤であってもよい。乾燥製剤の場合は、使用時に、注射用の精製水、生理食塩水、ブドウ糖液などの水性媒体に溶解して使用すればよい。液剤の場合は、そのまま投与してもよく、輸液、灌流液、腹膜透析液などに添加して使用してもよい。また、溶媒への溶解性や水性媒体への溶解性に問題のある場合や、徐放性の製剤を調製する場合には、両親媒性溶媒、油性基材や乳化剤などを使用して、有効成分の溶媒への溶解性を高めることも随意である。また、リポソームなどに封入して投与することも随意である。 In the case of parenteral preparations such as injection preparations, it is usually dissolved in an aqueous medium that does not contain pyrogen, depending on the target disease or symptom, and then intradermally, subcutaneously, intramuscularly, intracorporeally (intrathoracic) , Intraperitoneal, etc.), intravascular or intracerebral (including spinal cord), the preparation may be a dry preparation or a liquid preparation. In the case of a dry preparation, it may be used by dissolving it in an aqueous medium such as purified water for injection, physiological saline, and glucose solution at the time of use. In the case of a liquid preparation, it may be administered as it is, or it may be added to an infusion solution, a perfusion solution, a peritoneal dialysis solution, or the like. In addition, when there is a problem with solubility in a solvent or solubility in an aqueous medium, or when preparing a sustained-release preparation, it is effective to use an amphiphilic solvent, an oily base material, an emulsifier, etc. Increasing the solubility of the components in the solvent is optional. It is also optional to encapsulate and administer in liposomes.
　本発明でいう水性媒体とは、水を必須の要素とし、必要に応じて、これに、例えば、エタノール、プロパノール、イソプロパノールなどのアルコール類、アセトンなどのケトン類、ジエチルエーテルなどのエーテル類、ジメチルスルホキシド（以下、「ＤＭＳＯ」と略記する場合がある。）などの含硫化合物をはじめとする親水性有機溶剤の１又は複数を配合してなる水性媒体一般を意味する。この発明による液剤における水性溶剤としては、注射用精製水、生理食塩水、リンゲル液など、を単独で用いるか、あるいは、注射用精製水と、例えば、エタノール、プロパノール、イソプロパノール、ジエチルエーテル、ＤＭＳＯなどの生理学的に許容される親水性有機溶剤との混液を用いるのが望ましい。また、乳酸、塩酸、水酸化ナトリウム、水酸化カリウム、炭酸水素ナトリウムやリン酸緩衝液などのｐＨ調整剤を添加して、製剤化する化合物の最も溶解度の高いｐＨに調製することも随意である。 The aqueous medium referred to in the present invention has water as an essential element, and if necessary, for example, alcohols such as ethanol, propanol and isopropanol, ketones such as acetone, ethers such as diethyl ether, dimethyl It means a general aqueous medium comprising one or more hydrophilic organic solvents including sulfur-containing compounds such as sulfoxide (hereinafter sometimes abbreviated as “DMSO”). As the aqueous solvent in the liquid preparation according to the present invention, purified water for injection, physiological saline, Ringer's solution or the like is used alone, or purified water for injection and, for example, ethanol, propanol, isopropanol, diethyl ether, DMSO, etc. It is desirable to use a mixture with a physiologically acceptable hydrophilic organic solvent. It is also optional to add a pH adjuster such as lactic acid, hydrochloric acid, sodium hydroxide, potassium hydroxide, sodium bicarbonate or phosphate buffer to adjust the pH to the highest solubility of the compound to be formulated. .
　斯かる液剤の場合、使用する一般式１で表される化合物によっては、溶存酸素などにより不安定になる場合があるので、その場合は、例えば、該化合物溶液の溶存酸素濃度を低減させればよい。このような液状組成物は、通常、該化合物を水性媒体に溶解する工程と、該水性媒体をしてその常温常圧の大気環境下における酸素濃度を下回らせる工程とを経由する方法により調製することができる。これらの化合物を水性媒体に溶解するには、例えば、所定の量の化合物を適量の水性媒体へ添加し、必要に応じて、加熱・攪拌しながら溶解させた後、必要に応じて、化合物の濃度が所定のレベルになるまで水性媒体を追加すればよい。 In the case of such a liquid agent, depending on the compound represented by the general formula 1 used, it may become unstable due to dissolved oxygen or the like. In that case, for example, if the dissolved oxygen concentration of the compound solution is reduced, Good. Such a liquid composition is usually prepared by a method through a step of dissolving the compound in an aqueous medium and a step of lowering the oxygen concentration in the atmospheric environment at normal temperature and pressure using the aqueous medium. be able to. In order to dissolve these compounds in an aqueous medium, for example, a predetermined amount of a compound is added to an appropriate amount of an aqueous medium, dissolved as necessary with heating and stirring, and then, if necessary, An aqueous medium may be added until the concentration reaches a predetermined level.
　水性媒体の溶存酸素濃度を、常温常圧の大気環境下における濃度より低くするには、例えば、一般式１で表される化合物溶液を減圧下で調製し、保存するか、該化合物溶液に溶存する酸素を別の気体で置換するか、あるいは、該化合物溶液を脱酸素剤へ接触させる方法が好適である。液状組成物に溶解する酸素を別の気体で置換するには、液状組成物中で、例えば、窒素などの比較的不活性な気体か、あるいは、ネオン、アルゴン、クリプトン、キセノンなどの希ガスをバブリングさせればよい。脱酸素剤を用いて酸素濃度を下げるには、液状組成物へ、例えば、Ｌ－アスコルビン酸、Ｌ－アスコルビン酸ステアリン酸エステル、亜硫酸ナトリウム、亜硫酸水素ナトリウム、アルファチオグリセリン、エデト酸ナトリウム、塩酸システイン、クエン酸、大豆レシチン、チオグリコール酸ナトウム、チオリンゴ酸ナトリウム、ピロ亜硫酸ナトリウム、ブチルヒドロキシアニソールなどを適量添加すればよい。これらの方法は、化合物溶液に適用しても、化合物を溶解する前の水性媒体へ適用してもよい。この場合の水性媒体に溶存する酸素の濃度は、通常、０．４ｐｐｍ以下、望ましくは、０．１ｐｐｍ以下とすればよい。また、トコフェロール、カロチン、ヒスチジン、トリプトファン、チロシン、メチオニン、システイン、ドーパ、ルチン、ルチン誘導体、チオタウリン、ヒポタウリン、ビリルビン、コレステロール、キノリン、ケルセチン、カテキン、アントシアニン、チアミンなどのような一重項酸素消去活性を有する成分や、アルキルセルロース、カルボキビニルポリマーなどの造粘剤、トリトンＸ、ポリソルベイト、デオキシコール酸又はその塩、コール酸又はその塩などの界面活性剤を、本発明の有効成分として使用する化合物の安定化のため適量添加して、製剤を調製することも有利に実施できる。 In order to make the dissolved oxygen concentration of the aqueous medium lower than the concentration in the atmospheric environment at normal temperature and normal pressure, for example, the compound solution represented by the general formula 1 is prepared under reduced pressure and stored or dissolved in the compound solution. It is preferable to replace oxygen with another gas or to bring the compound solution into contact with an oxygen scavenger. In order to replace oxygen dissolved in the liquid composition with another gas, in the liquid composition, for example, a relatively inert gas such as nitrogen or a rare gas such as neon, argon, krypton, or xenon is used. Just bubbling. In order to reduce the oxygen concentration using an oxygen scavenger, the liquid composition is prepared by, for example, adding L-ascorbic acid, L-ascorbic acid stearate, sodium sulfite, sodium hydrogen sulfite, alphathioglycerin, sodium edetate, cysteine hydrochloride Citric acid, soybean lecithin, sodium thioglycolate, sodium thiomalate, sodium pyrosulfite, butylhydroxyanisole and the like may be added in appropriate amounts. These methods may be applied to the compound solution or to an aqueous medium before the compound is dissolved. In this case, the concentration of oxygen dissolved in the aqueous medium is usually 0.4 ppm or less, preferably 0.1 ppm or less. It also has singlet oxygen scavenging activity such as tocopherol, carotene, histidine, tryptophan, tyrosine, methionine, cysteine, dopa, rutin, rutin derivatives, thiotaurine, hypotaurine, bilirubin, cholesterol, quinoline, quercetin, catechin, anthocyanin, thiamine, etc. Of compounds using as active ingredients of the present invention, surfactants such as thickeners such as alkyl cellulose and carboxyl polymer, Triton X, polysorbate, deoxycholic acid or salts thereof, and cholic acid or salts thereof It is also possible to advantageously carry out the preparation by adding an appropriate amount for stabilization.
　斯くして得られた一般式１で表される化合物の溶液は、酸素を遮断し得る、用途に応じた適宜の容器へ封入した状態で保存すればよい。容器の材質としては、原理上、液状組成物を保持することができ、かつ、酸素を実質的に遮断し得るものであるかぎり、特に制限がないが、褐色ビンや褐色のアンプルのような遮光性の容器が望ましい。用途にもよるけれども、通常、ガラスアンプル、バイアル瓶などの容器へ液状組成物を分注前に濾過滅菌などの滅菌を行うか、分注して容器を封止した後、高圧滅菌や濾過滅菌などにより滅菌する。 The thus obtained solution of the compound represented by the general formula 1 may be stored in a state where it is sealed in an appropriate container that can block oxygen. The material of the container is not particularly limited as long as it can hold the liquid composition in principle and can substantially block oxygen, but it is light-shielded like a brown bottle or brown ampoule. Sex containers are desirable. Although it depends on the application, sterilization such as filtration sterilization is usually performed before dispensing the liquid composition into containers such as glass ampoules and vials, or after dispensing and sealing the container, high-pressure sterilization or filtration sterilization is performed. Sterilize by etc.
　また、本発明の抗神経変性疾患剤は、注射剤以外にも、ハップ剤や経肺用の吸飲噴霧剤などの形態で使用することもでき、皮下などの体内に埋め込む徐放製剤の形態で使用することもできる。また、神経変性疾患を発症したペットをはじめとするヒト以外の動物の治療や、神経変性症に伴う病態や神経機能障害の予防剤乃至治療剤として使用することも随意である。 In addition to the injection, the anti-neurodegenerative disease agent of the present invention can be used in the form of a haptic agent, a transpulmonary sucking and spraying agent, etc. Can also be used. It is also optional to treat animals other than humans, including pets that have developed neurodegenerative diseases, and to use as preventives or therapeutic agents for pathological conditions and neurological dysfunction associated with neurodegenerative diseases.
　このようにして製造される本発明の抗神経変性疾患剤は、長期間連用しても、重篤な副作用もなく安全な製剤である。 The anti-neurodegenerative disease agent of the present invention thus produced is a safe preparation without serious side effects even when used for a long time.
　本発明の抗神経変性疾患剤は、対象とする神経変性疾患、その病態や症状に応じて、毎日乃至１日以上の間隔をおいて、一日に一回乃至複数回に分けて一日あたりの所定量を投与すればよい。一日当たりの投与量は、所期の作用・効果が得られる量であれば特に制限はなく、通常、静脈内投与（点滴を含む）、皮下乃至腹腔内投与の場合、一般式１で表される化合物を合計で、０．０１ｍｇ／ｋｇ・体重／日以上が望ましく、０．１乃至２０ｍｇ／ｋｇ・体重／日がより望ましく、０．５乃至５ｍｇ／ｋｇ・体重／日が特に望ましい。５０ｍｇ／ｋｇ・体重／日以上投与しても、その投与量に見合うほどの効果の増強は認められない場合がある。なお、本発明の抗神経変性疾患剤のラジカルスカベンジャーとしての機能を期待して投与する場合には、上記投与量よりも増量して投与することも随意である。また、本発明の抗神経変性疾患症剤の投与期間は、対象とする疾患、病態乃至症状に応じて調製すればよく、急性の疾患の場合には症状が改善乃至消失するまで投与すればよく、認知症などのような慢性疾患の場合には、症状の改善乃至消失が認められた場合でも、投与を継続することが望ましい。 The anti-neurodegenerative disease agent of the present invention is divided into once or multiple times per day at intervals of daily to 1 day or more depending on the target neurodegenerative disease, its pathology and symptoms. May be administered in a predetermined amount. The daily dose is not particularly limited as long as the desired action and effect can be obtained, and is generally expressed by the general formula 1 in the case of intravenous administration (including infusion) and subcutaneous to intraperitoneal administration. In total, 0.01 mg / kg · body weight / day or more is desirable, 0.1 to 20 mg / kg · body weight / day is more desirable, and 0.5 to 5 mg / kg · body weight / day is particularly desirable. Even when administered at 50 mg / kg · body weight / day or more, there is a case where the enhancement of the effect corresponding to the dose is not observed. In addition, when administering in anticipation of the function as a radical scavenger of the anti-neurodegenerative disease agent of the present invention, it is also optional to increase the dose above the above dose. In addition, the administration period of the anti-neurodegenerative disease agent of the present invention may be prepared according to the target disease, condition or symptom, and may be administered until the symptom is improved or disappeared in the case of an acute disease. In the case of chronic diseases such as dementia, it is desirable to continue administration even if improvement or disappearance of symptoms is observed.
　本発明の抗神経変性疾患剤は、脳や神経細胞を傷害因子から保護して変性を抑制すると共に、神経細胞を活性化し、神経突起の伸展促進や萎縮の抑制、神経細胞の生存延長や変性を抑制することもできるので、神経変性疾患、とりわけ、中枢神経の変性に起因する疾患を治療することができる。神経変性疾患とは、神経細胞（中枢神経（例えば、脳神経、脊髄神経など）及び／又は末梢神経（例えば、自律神経系（例えば、交感神経、副交感神経など）、運動神経系、知覚神経系））の変性を伴う疾患全てを包含し、その病因によって限定されるものではない。具体的には、一般的に神経変性疾患とされている疾患であればよく、例えば、パーキンソン病、パーキンソン症候群、線条体黒質変性症、ハンチントン病、舞踏病－無定位運動症、進行性核上麻痺、びまん性レビー小体病、大脳皮質基底核変性症、アルツハイマー病、老年性認知症、ピック病、前頭側頭葉型認知症、家族性認知症、脊髄小脳変性症（例えば、オリーブ橋小脳萎縮症、晩発性小脳皮質萎縮症、家族性脊髄小脳失調症（例えば、マッカードジョセフ病など）、歯状核赤核淡蒼球ルイ体萎縮症、家族性痙性対麻痺、フリードライヒ病など）、運動神経病（例えば、筋萎縮性側索硬化症、家族性筋萎縮性側索硬化症など）、脱髄性疾患（例えば、多発性硬化症、汎発硬化症、急性散在性脳脊髄炎、急性小脳炎、横断性脊髄炎、ギラン・バレー症候群など）、代謝性脳疾患、先天性及び遺伝性疾患（神経系リソソーム蓄積症など）などだけでなく、脳血管障害（例えば、脳卒中、脳梗塞（例えば、脳血栓、脳塞栓など）、低温療法などの治療中の脳血管障害、低酸素性虚血性脳障害、一過性脳虚血発作、再灌流障害、脳出血（例えば、高血圧性脳内出血、クモ膜下出血など）など）・脳腫瘍（例えば、星状膠細胞腫、脳膿瘍など）・血液量減少性ショック・外傷性ショック・頭部損傷及び／又は脳脊髄外傷（例えば、脳挫傷・貫入・せん断・圧迫・裂傷、分娩時外傷、乳児むち打ち揺さぶり症候群など）に伴う神経機能障害、感染症に伴う脳脊髄疾患（例えば、髄膜炎、インフルエンザ脳症、クロイツフェルト－ヤコブ病、エイズ脳症による認知症など）、毒物（ヒ素、カドミウム、有機水銀、サリン、ソマン、タブン、ＶＸガスなど）・放射線などによる神経機能障害、精神疾患（例えば、神経症、心身症、不安、統合失調症、躁うつ病など）、てんかん、メージ症候群、ジストニア、ダウン症、睡眠障害（例えば、過眠、ナルコレプシー、睡眠時無呼吸症候群など）なども含まれる。 The anti-neurodegenerative disease agent of the present invention protects the brain and nerve cells from injury factors and suppresses degeneration, activates nerve cells, promotes neurite extension and suppresses atrophy, prolongs survival and degeneration of nerve cells Therefore, it is possible to treat neurodegenerative diseases, particularly diseases caused by degeneration of the central nervous system. A neurodegenerative disease is a nerve cell (central nervous system (for example, cranial nerve, spinal nerve), and / or peripheral nerve (for example, autonomic nervous system (for example, sympathetic nerve, parasympathetic nerve)), motor nervous system, sensory nervous system) ) Are included, and are not limited by the etiology. Specifically, any disease that is generally regarded as a neurodegenerative disease may be used. For example, Parkinson's disease, Parkinson's syndrome, striatal nigra degeneration, Huntington's disease, chorea-ataxia, progressive Supranuclear paralysis, diffuse Lewy body disease, basal ganglia degeneration, Alzheimer's disease, senile dementia, Pick's disease, frontotemporal lobar dementia, familial dementia, spinocerebellar degeneration (eg olive Bridge cerebellar atrophy, late cerebellar cortical atrophy, familial spinocerebellar ataxia (eg, Maccard Joseph disease, etc.), dentate nucleus erythrocytic Ryukyu atrophy, familial spastic paraplegia, Friedreich Disease), motor neuropathy (eg, amyotrophic lateral sclerosis, familial amyotrophic lateral sclerosis, etc.), demyelinating diseases (eg, multiple sclerosis, multiple sclerosis, acute disseminated) Encephalomyelitis, acute encephalitis, transverse myelitis, Guillain Valley syndrome, etc.), metabolic brain disease, congenital and hereditary diseases (neural lysosomal storage disease, etc.) as well as cerebrovascular disorders (eg, stroke, cerebral infarction (eg, cerebral thrombus, cerebral embolism, etc.), low temperature Cerebrovascular disorder, hypoxic ischemic brain disorder, transient ischemic attack, reperfusion injury, cerebral hemorrhage (eg, hypertensive intracerebral hemorrhage, subarachnoid hemorrhage, etc.) For example, astrocytoma, brain abscess, etc.) ・ blood loss shock, traumatic shock, head injury and / or cerebrospinal trauma (eg, brain contusion, penetration, shearing, compression, laceration, labor trauma, Nervous dysfunction associated with infant whiplash syndrome, cerebrospinal disease associated with infection (eg meningitis, influenza encephalopathy, Creutzfeldt-Jakob disease, dementia due to AIDS encephalopathy), poison (arsenic, cadence) , Organic mercury, sarin, soman, tabun, VX gas, etc.) ・ Neural dysfunction due to radiation, mental illness (eg, neurosis, psychosomatic disorder, anxiety, schizophrenia, manic depression, etc.), epilepsy, mage syndrome Dystonia, Down syndrome, sleep disorders (eg, hypersomnia, narcolepsy, sleep apnea syndrome, etc.).
　本発明の抗神経変性疾患剤の対象として好ましい該神経変性疾患は、例えば、パーキンソン病、パーキンソン症候群、ハンチントン病、アルツハイマー病、老年性認知症、脊髄小脳変性症、筋萎縮性側索硬化症、脱髄性疾患（例えば、多発性硬化症など）、脳血管障害（例えば、脳卒中、脳梗塞（例えば、脳血栓、脳塞栓など）、一過性脳虚血発作、脳出血（例えば、高血圧性脳内出血、クモ膜下出血など）など）・脳腫瘍・外傷性ショック・頭部損傷及び／又は脳脊髄外傷（例えば、脳挫傷など）に伴う神経機能障害、感染症に伴う脳脊髄疾患（例えば、髄膜炎、インフルエンザ脳炎・脳症、クロイツフェルト－ヤコブ病、エイズ脳症による認知症など）、てんかんなどの中枢神経系の神経変性に由来する疾患であり、より好ましくは、例えば、パーキンソン病、パーキンソン症候群、アルツハイマー病、筋萎縮性側索硬化症に伴う神経機能障害、感染症に伴う脳脊髄疾患（例えば、髄膜炎、インフルエンザ脳症、クロイツフェルト－ヤコブ病、エイズ脳症による認知症など）、てんかんなどであり、特に好ましくは、例えば、パーキンソン病、アルツハイマー病に伴う神経機能障害などである。 The preferred neurodegenerative disease as a subject of the anti-neurodegenerative disease agent of the present invention is, for example, Parkinson's disease, Parkinson's syndrome, Huntington's disease, Alzheimer's disease, senile dementia, spinocerebellar degeneration, amyotrophic lateral sclerosis, Demyelinating diseases (for example, multiple sclerosis), cerebrovascular disorders (for example, stroke, cerebral infarction (for example, cerebral thrombosis, cerebral embolism, etc.), transient cerebral ischemic attacks, cerebral hemorrhage (for example, hypertensive intracerebral hemorrhage) ), Brain tumors, traumatic shock, head injury and / or cerebrospinal trauma (eg, cerebral contusion), cerebrospinal disease (eg, meninges) Inflammation, influenza encephalitis / encephalopathy, Creutzfeldt-Jakob disease, dementia due to AIDS encephalopathy, etc.), diseases derived from central nervous system neurodegeneration such as epilepsy, more preferably For example, Parkinson's disease, Parkinson's syndrome, Alzheimer's disease, neurological dysfunction associated with amyotrophic lateral sclerosis, cerebrospinal disease associated with infection (eg, meningitis, influenza encephalopathy, Creutzfeldt-Jakob disease, AIDS encephalopathy Dementia and the like), epilepsy and the like, particularly preferably, for example, neurological dysfunction associated with Parkinson's disease and Alzheimer's disease.
　さらに、本発明の抗神経変性疾患剤は、神経細胞を活性化し、神経突起を伸展させ、シナプスの形成を促進するなどにより、神経機能障害をも治療することができる。対象となる神経機能障害とは、神経機能の障害であればどのようなものであってもよいが、例えば、認知機能障害、意識障害、両側性四肢麻痺、反対側片麻痺、交代性片麻痺、顔面神経麻痺、感覚障害、一過性失明（例えば、一過性黒内障など）、同名性半盲、めまい、眼振、複視、失語、耳鳴、昏睡などが挙げられる。特に好ましくは、前記神経変性疾患に伴うこれらの神経機能障害などが対象となる。前記の神経変性疾患に伴う神経機能障害、例えば、脳梗塞に伴う神経機能障害は、血管閉塞部位により様々であり、また障害されるレベルによっても症状が異なるが、主に上記の神経機能障害が見られる。また、脳梗塞における神経機能障害は、神経機能障害を検出する当技術分野で公知の様々な診断試験によってその有無を判断してもよい。該診断試験の具体的な例としては、例えば、アルツハイマー病などによる記憶や認知機能障害の評価に使用されている認知機能スコア（Ａｌｚｈｅｉｍｅｒ’ｓ　Ｄｉｓｅａｓｅ　Ａｓｓｅｓｓｍｅｎｔ　Ｓｃａｌｅ－ｃｏｇｎｉｔｉｖｅ　ｐａｒｔ；ＡＤＡＳ－ｃｏｇ）、臨床症状改善スコア（Ａｌｚｈｅｉｍｅｒ’ｓ　Ｄｉｓｅａｓｅ　Ｃｏｏｐｅｒａｔｉｖｅ　Ｓｔｕｄｙ－Ｃｌｉｎｉｃａｌ　Ｇｌｏｂａｌ　Ｉｍｐｒｅｓｓｉｏｎ　ｏｆ　Ｃｈａｎｇｅ；ＡＤＣＳ－ＣＧＩＣ）、ミニ精神状態試験（Ｍｉｎｉ－Ｍｅｎｔａｌ　Ｓｔａｔｅ　Ｅｘａｍｉｎａｔｉｏｎ；ＭＭＳＥ）、長谷川式評価法などをあげることができ、さらには、グラスゴーアウトカムスケール（Ｇｌａｓｇｏｗ　Ｏｕｔｃｏｍｅ　Ｓｃａｌｅ：ＧＯＳ）、グラスゴーコーマスケール（Ｇｌａｓｇｏｗ　Ｃｏｍａ　Ｓｃａｌｅ：ＧＣＳ）、ランキンスケール（Ｒａｎｋｉｎ　Ｓｃａｌｅ：ＲＳ）、改変ランキンスケール（ｍｏｄｉｆｉｅｄ　Ｒａｎｋｉｎ　Ｓｃａｌｅ：ｍＲＳ）、能力障害関連スケール（Ｄｉｓａｂｉｌｉｔｙ　Ｒａｔｉｎｇ　Ｓｃａｌｅ：ＤＲＳ）、及び、ＮＩＨ卒中スケール（ＮＩＨ　Ｓｔｒｏｋｅ　Ｓｃａｌｅ：ＮＩＨＳＳ）などの公知の方法を用いて行うことができる。これらの神経機能障害を検出する診断試験は、物理的な脳の異常を検出する試験方法、例えば、ＣＴスキャンや頭蓋内圧の測定などと適宜組み合わせておこなってもよい。 Furthermore, the anti-neurodegenerative disease agent of the present invention can also treat nerve dysfunction by activating nerve cells, extending neurites, and promoting synapse formation. The target neurological dysfunction may be any neurological dysfunction, such as cognitive dysfunction, consciousness disorder, bilateral quadriplegia, contralateral hemiplegia, alternating hemiplegia. Facial paralysis, sensory impairment, transient blindness (eg, transient cataract), homonymous half-blindness, dizziness, nystagmus, double vision, aphasia, tinnitus, coma and the like. Particularly preferred are those neurological dysfunctions associated with the neurodegenerative diseases. The neurological dysfunction associated with the above-mentioned neurodegenerative diseases, for example, neurological dysfunction associated with cerebral infarction varies depending on the site of vascular occlusion, and the symptoms vary depending on the level to be impaired. It can be seen. In addition, the presence or absence of neurological dysfunction in cerebral infarction may be determined by various diagnostic tests known in the art for detecting neurological dysfunction. Specific examples of the diagnostic test include, for example, a cognitive function score (Alzheimer's Dissease Assessment-cognitive part; ADAS-cog) used for evaluation of memory and cognitive impairment due to Alzheimer's disease, clinical symptoms, and the like. Improvement score (Alzheimer's Dissease Cooperative Perspective-Clinical Global Impression of Change; ADCS-CGIC), Mini Mental State Exam (Mini-Mental State Examination; MMSE) Outcome scale (Glasgow Outcome Scale: GOS), glass -Comascale (Glasgow Coma Scale: GCS), Rankin Scale (Rakin Scale: RS), Modified Rankin Scale (Modified Rankin Scale: mRS), Disability-related Scale (Disability Rating Scale: DRS), and NS : NIHSS) or the like. These diagnostic tests for detecting neurological dysfunction may be performed in appropriate combination with a test method for detecting physical brain abnormalities, for example, a CT scan or intracranial pressure measurement.
　したがって、本発明の抗神経変性疾患剤は、神経細胞保護剤、神経細胞活性化剤、神経突起伸展促進剤、神経突起萎縮抑制剤、プルキンエ細胞変性・脱落抑制剤、神経変性疾患に伴う病態の治療剤、神経機能障害治療剤などとして有利に使用することができる。本発明でいう神経変性疾患や神経機能障害の治療とは、神経変性に起因する病態や機能障害を治癒の方向へ導く、いわゆる治療に加え、悪化を抑制し病態の進行をとどめる進展防止、さらには疾患の発症そのものの予防も含む。 Therefore, the anti-neurodegenerative disease agent of the present invention includes a neuron protective agent, a neuron activator, a neurite extension promoter, a neurite atrophy inhibitor, a Purkinje cell degeneration / dropout inhibitor, and a pathological condition associated with a neurodegenerative disease. It can be advantageously used as a therapeutic agent, a neurological dysfunction therapeutic agent or the like. The treatment of neurodegenerative diseases and neurological dysfunctions as used in the present invention refers to the pathology and dysfunction caused by neurodegeneration in the direction of healing, in addition to so-called treatment, prevention of progression that suppresses progression and stops progression of the disease, Also includes the prevention of the onset of the disease itself.
　さらに、本発明の抗神経変性疾患剤は、ヒドロキシラジカルをはじめとするフリーラジカルを低減することができるので、脳内の神経や血管などの組織はいうまでもなく、脳以外の血管や臓器における、虚血後の再灌流、炎症性疾患（免疫疾患、アレルギー、腫瘍などを含む）、感染症、薬物、放射線或いは物理的な刺激などにより発生するフリーラジカルや過酸化脂質に起因するといわれている各種疾患や病態の予防剤、治療剤として有利に利用することができる。より具体的には、例えば、上記神経変性疾患の予防・治療剤としてだけでなく、脳保護剤、脳（神経細胞、血管内皮細胞）の酸化的障害抑制剤、虚血性脳障害抑制剤、脳梗塞巣進展抑制剤、脳浮腫抑制剤、遅発性神経死抑制剤、脳機能正常化剤、酸化ストレス抑制剤、抗潰瘍剤、血糖上昇抑制剤、白内障や角膜障害などの眼性疾患の予防・治療剤、移植臓器保存剤や移植組織（皮膚を含む）・臓器の壊死防止剤、急性腎不全・薬物などによる腎障害、皮膚組織障害、肺障害、肝繊維化、化学物質、エンドトキシン、熱傷などによる皮膚組織の機能の障害、虚血などによる肝障害、脊髄損傷、動脈などの血管壁障害、心筋などの筋肉障害、尿細管間質障害などの各種臓器の障害の治療・予防剤、放射線障害予防剤・治療剤、抗腫瘍剤、腫瘍転移抑制剤、細胞障害マーカー抑制剤、心筋炎、膵臓炎、腸炎、関節、アレルギーをはじめとする各種組織や臓器の炎症性疾患やそれに伴う組織障害の予防・治療剤、視細胞障害、視神経使用外、網膜疾患、聴覚細胞障害、聴覚神経障害などの感覚細胞、感覚神経或いは感覚器の障害の抑制剤、農薬や有機溶媒などによる薬物中毒の予防・治療剤、カルシウム・ナトリウム交換系阻害剤、疼痛や掻痒の予防・治療剤、プロテインキナーゼ刺激剤、ミトコンドリア脳筋症予防・治療剤、動脈閉塞・狭窄予防・治療剤、血液脳関門破綻抑制剤、薬物依存症治療剤、アポトーシス抑制剤、臓器や組織における過酸化脂質生成抑制剤、ヒドロキシラジカル、ペルオキシラジカル、ＮＯラジカルなどのラジカルスカベンジャーなどとしても有利に利用することができ、これらの障害に伴う、機能障害や臨床症状の予防・治療剤としても、当然、使用することができる。また、本発明の抗神経変性疾患剤は、アミロイドβペプチド凝集阻害剤、アミロイドβペプチド傷害抑制剤、コリンエステラーゼ活性阻害剤、セリン／スレオニンキナーゼ（Ａｋｔ）活性化剤、ホスファチヂルイノシトール（３，４，５）３リン酸キナーゼ（ＰＩ３Ｋ）－セリン／スレオニンキナーゼ（Ａｋｔ）カスケード活性化剤、サイクリックＡＭＰ濃度上昇促進剤、ＳＡＰＫ／ＪＮＫリン酸化抑制剤などとして利用することも随意である。 Furthermore, since the anti-neurodegenerative disease agent of the present invention can reduce free radicals including hydroxy radicals, it is not limited to tissues such as nerves and blood vessels in the brain, but also in blood vessels and organs other than the brain. It is said to be caused by reperfusion after ischemia, inflammatory diseases (including immune diseases, allergies, tumors, etc.), infections, drugs, radiation, or lipid peroxides generated by physical stimulation. It can be advantageously used as a prophylactic or therapeutic agent for various diseases and conditions. More specifically, for example, not only as a prophylactic / therapeutic agent for the above-mentioned neurodegenerative diseases, but also as a brain protective agent, a brain (nerve cell, vascular endothelial cell) oxidative disorder inhibitor, an ischemic brain disorder inhibitor, a brain Infarct development inhibitor, brain edema inhibitor, delayed neuronal death inhibitor, brain function normalizer, oxidative stress inhibitor, anti-ulcer agent, blood sugar elevation inhibitor, prevention of ocular diseases such as cataract and corneal disorder・ Therapeutic agents, transplanted organ preservatives and transplanted tissues (including skin), organ necrosis preventive agents, acute renal failure / drug damage caused by drugs, skin tissue disorders, lung disorders, liver fibrosis, chemicals, endotoxin, burns Therapeutic / preventive agents for various organ disorders such as impaired skin tissue function, liver damage caused by ischemia, spinal cord injury, arterial and other vascular wall disorders, myocardial and other muscle disorders, and tubulointerstitial disorders Disorder prevention / treatment agent, antitumor agent, tumor metastasis Anti-inflammatory agent, cytopathic marker inhibitor, myocarditis, pancreatitis, enteritis, joints, prophylactic / therapeutic agents for various tissue and organ inflammatory diseases such as allergies and associated tissue disorders, photoreceptor cell damage, non-use of optic nerve , Sensory cells such as retinal diseases, auditory cell disorders and auditory neuropathies, inhibitors of sensory nerve or sensory organ disorders, preventive / therapeutic agents for drug addiction with agricultural chemicals and organic solvents, calcium / sodium exchange inhibitor, pain And pruritus prevention / treatment agent, protein kinase stimulant, mitochondrial encephalomyopathy prevention / treatment agent, arterial occlusion / stenosis prevention / treatment agent, blood-brain barrier disruption inhibitor, drug dependence treatment agent, apoptosis inhibitor, organ and It can also be used advantageously as a lipid scavenger for tissues such as lipid peroxide production inhibitors, hydroxy radicals, peroxy radicals, and NO radicals. Can be, associated with these disorders, as an agent for preventing or treating dysfunction and clinical symptoms, of course, it can be used. Further, the anti-neurodegenerative disease agent of the present invention includes an amyloid β peptide aggregation inhibitor, an amyloid β peptide injury inhibitor, a cholinesterase activity inhibitor, a serine / threonine kinase (Akt) activator, a phosphatidylinositol (3,4). (5) Triphosphate kinase (PI3K) -serine / threonine kinase (Akt) cascade activator, cyclic AMP concentration increase promoter, SAPK / JNK phosphorylation inhibitor, etc. are also optional.
　以下、実験により本発明をさらに詳細に説明する。 Hereinafter, the present invention will be described in more detail by experiments.
＜実験１：神経細胞に対する傷害に及ぼすペンタメチン系シアニン色素の影響＞
　神経細胞は、栄養飢餓や活性酸素による傷害に対して非常に脆弱なことが知られている。この神経細胞の特性は、アルツハイマー病やハンチントン病をはじめとする神経変性疾患で認められる神経細胞死の原因と考えられている。そこで、細胞傷害因子の神経細胞に対する傷害性に及ぼすペンタメチン系シアニン色素の影響を調べる試験を以下のように実施した。 <Experiment 1: Effect of Pentamethine Cyanine Dye on Injury to Neurons>
Neurons are known to be very vulnerable to nutrient starvation and active oxygen injury. This characteristic of nerve cells is thought to be the cause of nerve cell death observed in neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. Then, the test which investigates the influence of the pentamethine type | system | group cyanine pigment | dye on the cytotoxicity with respect to the nerve cell of a cytotoxic factor was implemented as follows.
＜試験標品＞
　試験には、化学式２で表される化合物（株式会社林原生物化学研究所製造、「ＮＫ－４」）を試験標品として使用した。ＮＫ－４は水に難溶性のため、ＤＭＳＯ（ＳＩＧＭＡ社販売、商品番号「Ｄ８４１８」）に５ｍｇ／ｍｌの濃度で溶解した後、膜濾過（Ｍｉｌｌｉｐｏｒｅ社販売、商品名「Ｍｉｌｌｅｘ－ＬＧ　ＳＬＬＧ０２５ＳＳ」、ＤＭＳＯ耐性膜使用）し、ダルベッコのＭＥＭ培地（日水製薬株式会社販売、以下、「Ｄ－ＭＥＭ培地」と略記する。）でさらに希釈して、試験に供した。なお、ＤＭＳＯに溶解した試験標品を、Ｄ－ＭＥＭ培地で、試験に使用する濃度に希釈した場合、それに含まれる濃度のＤＭＳＯは、以下の各試験系に影響しないことを予め確認した。なお、以下の実験で用いたシアニン色素はいずれも株式会社林原生物化学研究所で合成したものを用いた。 <Test specimen>
In the test, a compound represented by Chemical Formula 2 (manufactured by Hayashibara Biochemical Laboratories, Inc., “NK-4”) was used as a test sample. Since NK-4 is hardly soluble in water, it is dissolved in DMSO (SIGMA, product number “D8418”) at a concentration of 5 mg / ml, and then membrane filtered (Millipore, product name “Millex-LG SLLG025SS”, DMSO resistant membrane was used), and further diluted with Dulbecco's MEM medium (sold by Nissui Pharmaceutical Co., Ltd., hereinafter abbreviated as “D-MEM medium”), and subjected to the test. When a test sample dissolved in DMSO was diluted with D-MEM medium to a concentration used for the test, it was confirmed in advance that the concentration of DMSO contained therein did not affect the following test systems. The cyanine dyes used in the following experiments were all synthesized by Hayashibara Biochemical Laboratories.
＜細胞傷害抑制作用の測定方法＞ <Method for measuring cytotoxicity-inhibiting action>
＜栄養飢餓による細胞傷害に及ぼすＮＫ－４の影響＞
　ヒトの神経細胞変性の研究に好適なモデルとして利用されている、ラット副腎褐色細胞腫由来のＰＣ－１２細胞のＮＧＦ（神経増殖因子）高感受性株（以下、「ＰＣ１２－ＨＳ細胞」という。ヒューマンサイエンス研究資源バンクより入手）を使用した。栄養飢餓環境として血清除去培地による培養をおこなった。ＰＣ１２－ＨＳ細胞は、凍結保存した細胞を解凍して、１０容積％ウシ胎仔血清（ＦＢＳ）加Ｄ－ＭＥＭ培地を用いて培養して試験に供した。試験に用いる細胞は、常法により、０．２５質量％トリプシン溶液を用いて剥離し、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈して、コラーゲンコートした９６ウエルプレート（ファルコン社販売、商品名「マイクロテストプレート・細胞培養用、平底」）に、５×１０３個／１００μｌ／ウエルになるように播種した。２４時間後に、培養上清を除去後、ＦＢＳを含まないＤ－ＭＥＭ培地で希釈し、表１に示す終濃度の２倍に調整したＮＫ－４のいずれかを１００μｌ／ウエル添加し、３日間培養した。３日目に培養上清を除去し、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で、１０質量／容積％に希釈したＡｌａｍａｒ　ｂｌｕｅ（Ｔｒｅｋ　Ｄｉａｇｎｏｓｔｉｃ社販売）溶液を、２００μｌ／ウエル添加し、６時間培養し、蛍光プレートリーダー（日本モレキュラーディバイス株式会社販売、商品名「ＳｐｅｃｔｒａＭａｘ　Ｇｅｍｉｎｉ　ＨＹ」）で５４４－５９０ｎｍの蛍光強度を測定した。対照として、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地（ＮＫ－４無添加）で細胞を培養し、同様に、Ａｌａｍａｒ　ｂｌｕｅ（Ｔｒｅｋ　Ｄｉａｇｎｏｓｔｉｃ社販売）溶液を加えて培養後、各ウエルの蛍光強度を測定した。対照の蛍光強度を１００％とした時の相対値を求め、各ウエルの細胞の生存率（％）として表１に併せて示す。なお、本試験及び以下の試験では、ＰＣ１２－ＨＳ細胞は、３７℃、５容積％ＣＯ２の条件下で、インキュベーター内で培養した。 <Influence of NK-4 on cell damage caused by nutrient starvation>
NGF (Nerve Growth Factor) hypersensitive strain of PC-12 cells derived from rat adrenal pheochromocytoma (hereinafter referred to as “PC12-HS cells”), which is used as a suitable model for studying human neuronal degeneration. Obtained from the Science Research Resource Bank). Cultivation was performed using a serum-removed medium as a nutrient starvation environment. PC12-HS cells were thawed from cryopreserved cells and cultured in 10% by volume fetal bovine serum (FBS) -added D-MEM medium for testing. Cells used in the test were detached by a conventional method using a 0.25% by mass trypsin solution, diluted with 10% by volume FBS-added D-MEM medium, and coated with a collagen-coated 96-well plate (available from Falcon, trade name). “Micro test plate / cell culture, flat bottom”) was seeded at 5 × 10 3 cells / 100 μl / well. After 24 hours, the culture supernatant was removed, diluted with D-MEM medium containing no FBS, and 100 μl / well of NK-4 adjusted to twice the final concentration shown in Table 1 was added for 3 days. Cultured. On day 3, the culture supernatant was removed, and Alamar blue (Trek Diagnostics) solution diluted to 10% by mass with 10% FBS-added D-MEM medium was added at 200 μl / well and cultured for 6 hours. Then, the fluorescence intensity at 544-590 nm was measured with a fluorescence plate reader (trade name “SpectraMax Gemini HY”, sold by Nippon Molecular Devices Co., Ltd.). As a control, cells were cultured in D-MEM medium supplemented with 10% by volume FBS (without NK-4). Similarly, after adding Alamar blue (Trek Diagnostics) solution, the fluorescence intensity of each well was measured. did. The relative value when the fluorescence intensity of the control is taken as 100% is determined and shown in Table 1 as the cell viability (%) in each well. In this test and the following tests, PC12-HS cells were cultured in an incubator under conditions of 37 ° C. and 5% by volume CO 2 .
＜過酸化水素による細胞傷害に及ぼすＮＫ－４の影響＞
　上記と同様に培養したＰＣ１２－ＨＳ細胞を、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈して、コラーゲンコートした９６ウエルプレートに、２×１０４個／１００μｌ／ウエルで播種して２４時間培養した。その後、８００μＭ過酸化水素水（和光純薬工業株式会社販売）を５０μｌ／ウエル（終濃度２００μＭ）と、表１に示す終濃度の４倍の濃度に１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈したＮＫ－４とを、各々５０μｌ／ウエル（ＮＫ－４の終濃度５ｎｇ乃至５０，０００ｎｇ／ｍｌ）を同時に添加して、インキュベーター内で２時間培養後、２５容積％グルタルアルデヒド（和光純薬工業株式会社販売）を２０μｌ／ウエル（終濃度２０容積％）添加して細胞を固定した。０．０５質量％メチレンブルー（和光純薬工業株式会社販売）を１００μｌ／ウエル加えて、ダイアップテイク法で、常法により、各ウエルの吸光度を測定した。対照として過酸化水素及びＮＫ－４を添加しなかった以外は同様に培養を行い、メチレンブルーを加えて吸光度を測定した。対照の細胞数（吸光度）を１００（％）とした時の相対値を求めて各ウエルの細胞の生存率（％）として表１に併せて示す。 <Influence of NK-4 on cell injury caused by hydrogen peroxide>
PC12-HS cells cultured in the same manner as above were diluted with 10% by volume FBS-added D-MEM medium, seeded at 2 × 10 4 cells / 100 μl / well in a collagen-coated 96-well plate, and cultured for 24 hours. did. Then, dilute 800 μM hydrogen peroxide solution (available from Wako Pure Chemical Industries, Ltd.) with 50 μl / well (final concentration 200 μM) and 10% FBS-added D-MEM medium to a concentration 4 times the final concentration shown in Table 1. 50 μl / well of each of NK-4 was added simultaneously (final concentration of NK-4 from 5 ng to 50,000 ng / ml), cultured in an incubator for 2 hours, and then 25 vol% glutaraldehyde (Wako Pure Chemical Industries, Ltd.). The cells were fixed by adding 20 μl / well (final concentration 20% by volume). 0.05 μm methylene blue (available from Wako Pure Chemical Industries, Ltd.) was added at 100 μl / well, and the absorbance of each well was measured by a conventional method using a die-up take method. As a control, culture was carried out in the same manner except that hydrogen peroxide and NK-4 were not added, and methylene blue was added to measure the absorbance. The relative value when the number of cells of the control (absorbance) is 100 (%) is determined and shown in Table 1 as the cell viability (%) of each well.
＜アミロイドβフラグメントによる細胞傷害に及ぼすＮＫ－４の影響＞
　アルツハイマー病における神経細胞死の主要原因の一つと考えられているアミロイドβペプチド（ヒト由来）のアミノ末端から２５乃至３５番目に相当するアミノ酸配列を有するペプチドフラグメント（ＡｎａＳｐｅｃ社販売、以下、「アミロイドβフラグメント」という。）（配列表における配列番号１のアミノ酸配列を有するペプチド）を、リン酸緩衝生理食塩水（ＰＢＳ）で希釈して２ｍＭの濃度に調整し、使用前に６時間、３７℃でエイジングさせ、フラグメントを凝集させて、細胞毒性を上昇させたものを使用した。上記と同様に培養したＰＣ１２－ＨＳ細胞を、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈して、コラーゲンコートした９６ウエルプレートに、５×１０３個／１００μｌ／ウエルになるように播種し、２４時間培養後に上清を除去して、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈したアミロイドβフラグメント溶液を５０μｌ／ウエル（アミロイドβフラグメントの終濃度５０μＭ）と、ＮＫ－４溶液を５０μｌ／ウエル（終濃度４０乃至５０００ｎｇ／ｍｌ）添加して３日間培養した。３日目に培養上清を除去し、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で、１０質量％濃度に希釈したＡｌａｍａｒ　ｂｌｕｅ（Ｔｒｅｋ　Ｄｉａｇｎｏｓｔｉｃ社販売）溶液を、２００μｌ／ウエル添加し、６時間培養し、蛍光プレートリーダーで５４４－５９０ｎｍの蛍光強度を測定した。対照として、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地のみで細胞を培養（アミロイドβフラグメント及びＮＫ－４の両方とも無添加）し、同様にＡｌａｍａｒ　ｂｌｕｅ溶液を加えて、蛍光強度を測定した。対照の蛍光強度を１００％とする相対値を求めて、各ウエルの細胞の生存率（％）として表１に併せて示す。また、別途、前記と同じ条件で培養した細胞の培養上清を除去後、ＰＢＳで希釈した１容積％グルタルアルデヒドを１００μｌ／ウエル添加して３０分間固定した後、１ｍＭヘキスト３３２５８色素（ＳＩＧＭＡ社販売）で５分間染色した後、位相差顕微鏡下及び蛍光顕微鏡下で、一視野に約１００個の細胞を含む倍率で細胞を観察し、細胞をカウントして、同一視野内の全細胞中に占めるアポトーシスを起こしている細胞の占有率（％）を計算した結果を、表１に併せて示す。なお、アポトーシスを起こしている細胞の判定は、細胞の核の断片化乃至核内のクロマチンの凝集を指標とした。 <Influence of NK-4 on cell injury by amyloid β fragment>
Peptide fragments having an amino acid sequence corresponding to the 25th to 35th amino acids from the amino terminus of amyloid β peptide (human origin), which is considered to be one of the main causes of neuronal cell death in Alzheimer's disease (sold by AnaSpec, hereinafter “Amyloid β Fragment ") (a peptide having the amino acid sequence of SEQ ID NO: 1 in the sequence listing) was diluted with phosphate buffered saline (PBS) to a concentration of 2 mM, and 6 hours at 37 ° C before use. Aged and aggregated fragments were used to increase cytotoxicity. PC12-HS cells cultured in the same manner as above were diluted with 10% by volume FBS-added D-MEM medium, and seeded in a 96-well plate coated with collagen at 5 × 10 3 cells / 100 μl / well. After culturing for 24 hours, the supernatant was removed, and 50 μl / well of amyloid β fragment solution diluted with 10% by volume FBS-added D-MEM medium (final concentration of amyloid β fragment 50 μM) and NK-4 solution were 50 μl / well. (Final concentration 40 to 5000 ng / ml) was added and cultured for 3 days. On the third day, the culture supernatant was removed, and Alamar blue (Trek Diagnostics) solution diluted to 10% by mass with 10% by volume FBS-added D-MEM medium was added at 200 μl / well and cultured for 6 hours. The fluorescence intensity at 544-590 nm was measured with a fluorescence plate reader. As a control, cells were cultured only in D-MEM medium supplemented with 10% by volume FBS (no addition of both amyloid β fragment and NK-4), and Alamar blue solution was added in the same manner, and fluorescence intensity was measured. Relative values with the fluorescence intensity of the control as 100% were determined and shown in Table 1 as the cell viability (%) in each well. Separately, after removing the culture supernatant of cells cultured under the same conditions as described above, 100 μl / well of 1% by volume glutaraldehyde diluted with PBS was added and fixed for 30 minutes, and then 1 mM Hoechst 33258 dye (sold by SIGMA) ) For 5 minutes, and then, under a phase contrast microscope and a fluorescence microscope, the cells are observed at a magnification containing about 100 cells in one field, and the cells are counted and occupied in all cells in the same field The results of calculating the occupancy ratio (%) of cells undergoing apoptosis are also shown in Table 1. The determination of cells undergoing apoptosis was based on cell nucleus fragmentation or aggregation of chromatin in the nucleus.
Figure JPOXMLDOC01-appb-T000024
Figure JPOXMLDOC01-appb-T000024
　表１から明らかなように、ＮＫ－４は、ＰＣ１２－ＨＳ細胞に対する栄養飢餓、過酸化水素傷害、アミロイドβフラグメントによる傷害の何れに対しても、各々の細胞傷害因子に対する有効濃度に差はあるものの、神経細胞に対する保護作用を有することが明らかとなった。表１に示す３種類の細胞傷害因子に対する保護作用が発揮されるＮＫ－４の濃度を比較すると、アミロイドβフラグメントの傷害に対する保護作用は４０ｎｇ／ｍｌから発揮されているのに対して、栄養飢餓傷害では５００ｎｇ／ｍｌ、過酸化水素傷害では５，０００ｎｇ／ｍｌの濃度が各々必要であることが判明した。傷害の発生する速度を見ると、過酸化水素が最も早く、アポトーシス誘導を伴うアミロイドβフラグメントが最も遅いと考えられるので、これらの細胞傷害因子に対するＮＫ－４による細胞保護作用は、傷害の発生する速度が速い程、高い濃度を必要とすることを物語っている。なお、具体的なデータは示さないが、濃度が５０，０００ｎｇ／ｍｌ以下のＮＫ－４水溶液は過酸化水素に対する消去能は有していないので、ＮＫ－４はペルオキシラジカルルやヒドロキシラリジカルなどのフリーラジカルの消去能も有しているものの、この試験系における過酸化水素による細胞傷害に対する細胞保護作用は、直接過酸化水素を消去する作用によるものではなく、細胞側に作用して、細胞死を抑制していると判断した。また、ヘキスト染色像より算出したアポトーシスを起こした細胞の占有率は、アミロイドβフラグメントを添加した場合（ＮＫ－４濃度０ｎｇ／ｍｌ）７２％となってアポトーシスが促進されたのに対して、ＮＫ－４（２００ｎｇ／ｍｌ）を添加した場合の占有率は１３％となって、対照のアポトーシスを起こした細胞の占有率（５％）にほぼ近い値となり、ＮＫ－４が、アミロイドβフラグメントにより誘導されるアポトーシスを抑制することが確認された。また、具体的なデータは示さないが、位相差顕微鏡観察でもアミロイドβフラグメントの添加により、細胞の凝集及び細胞死が認められたのに対して、ＮＫ－４の添加により細胞の凝集や細胞死が抑制されていることが確認された。さらに、ヘキスト染色像でも、アミロイドβフラグメントの添加により認められるアポトーシスによるクロマチンの凝集及び核の断片化が、ＮＫ－４を添加することにより、抑制されることが確認された。以上の結果は、ＮＫ－４が栄養飢餓傷害に対する神経細胞保護作用があることから神経栄養因子活性を有しているので、ＮＫ－４は、神経細胞保護剤や神経栄養因子として使用できることを物語っている。また、ＮＫ－４は、複数の細胞傷害因子に対して細胞を保護し、細胞死を抑制する作用を有しているので、神経変性抑制活性を有し、アミロイドβペプチドをはじめとする細胞傷害因子により引き起こされるアルツハイマー病などに代表されるヒトの神経変性疾患の有効な治療剤として利用できることを物語っている。また、ＮＫ－４はアポトーシス抑制剤としても利用できることを物語っている。 As is clear from Table 1, NK-4 has a difference in effective concentration for each cytotoxic factor against nutrient starvation, hydrogen peroxide damage, and amyloid β fragment damage to PC12-HS cells. However, it has been found to have a protective effect on nerve cells. Comparing the concentrations of NK-4 that exert the protective action against the three types of cytotoxic factors shown in Table 1, the protective action against the damage of the amyloid β fragment is exerted from 40 ng / ml, whereas the nutrient starvation It was found that a concentration of 500 ng / ml was required for injury and a concentration of 5,000 ng / ml was required for hydrogen peroxide injury. When looking at the rate at which injury occurs, hydrogen peroxide is the fastest and amyloid β fragment with apoptosis induction is considered to be the slowest, so the cytoprotective action by NK-4 against these cytotoxic factors causes injury. The higher the speed, the higher the concentration required. Although specific data is not shown, NK-4 aqueous solution having a concentration of 50,000 ng / ml or less does not have the ability to erase hydrogen peroxide. Although it also has the ability to scavenge free radicals in this test system, the cytoprotective action against cell damage by hydrogen peroxide in this test system is not due to the action of directly removing hydrogen peroxide, but acts on the cell side, Judged to suppress death. In addition, the occupancy ratio of the cells having undergone apoptosis calculated from Hoechst staining image was 72% when amyloid β fragment was added (NK-4 concentration 0 ng / ml), whereas apoptosis was promoted. -4 (200 ng / ml) was added, the occupancy was 13%, which was close to the occupancy (5%) of apoptotic cells in the control, and NK-4 was reduced by amyloid β fragment. It was confirmed to suppress the induced apoptosis. Although specific data are not shown, cell aggregation and cell death were observed by addition of amyloid β fragment in phase contrast microscopy, whereas cell aggregation and cell death were observed by addition of NK-4. Was confirmed to be suppressed. Furthermore, it was also confirmed in the Hoechst-stained image that the addition of NK-4 suppresses the aggregation of chromatin and the fragmentation of the nucleus due to apoptosis, which is recognized by the addition of amyloid β fragment. The above results indicate that NK-4 can be used as a neuroprotective agent or neurotrophic factor because it has neurotrophic factor activity because it has a neuroprotective action against nutrient starvation injury. ing. In addition, NK-4 protects cells against a plurality of cytotoxic factors and suppresses cell death. Therefore, NK-4 has neurodegeneration inhibitory activity and cytotoxicity including amyloid β peptide. It shows that it can be used as an effective therapeutic agent for human neurodegenerative diseases represented by factors such as Alzheimer's disease. It also shows that NK-4 can be used as an apoptosis inhibitor.
　なお、具体的なデータは示さないが、ＰＣ１２細胞に対する神経増殖因子（ＮＧＦ）の作用はＴｒｋＡに特異性の高いプロテインキナーゼ阻害剤であるＫ２５２ａにより阻害されることが報告されているので（Ｋａｓｅ　Ｈ．等、『Ｂｉｏｃｈｅｍｉｃａｌ　ａｎｄ　Ｂｉｏｐｈｙｓｉｃａｌ　Ｒｅｓｅａｒｃｈ　Ｃｏｍｍｕｎｉｃａｔｉｏｎｓ』、　第１４４巻、第３５－４０頁（１９８７年）参照）、ＮＫ－４がＮＧＦと同じ経路でＰＣ１２－ＨＳ細胞に作用しているかどうかを検討したところ、ＮＫ－４による、ＰＣ１２－ＨＳ細胞増殖促進作用や後述の神経突起伸展作用は、Ｋ２５２ａでは阻害されなかった。一方、ＮＫ－４の作用は、ホスファチヂルイノシトール（３，４，５）３リン酸キナーゼ（ＰＩ３Ｋ）を阻害することで、ホスファチヂルイノシトール（３，４，５）３リン酸の産生を抑制し、最終的に、細胞の生存や増殖に主要な働きをするセリン／スレオニンキナーゼ（Ａｋｔ）の活性化を抑制するＬＹ２９４００２（Ｖｌａｈｏｓ　Ｃ．等、『Ｊｏｕｒｎａｌ　ｏｆ　Ｂｉｏｌｏｇｉｃａｌ　Ｃｈｅｍｉｓｔｒｙ』、第２６９巻、第５２４１乃至５２４８頁（１９９４年）参照）で阻害されること、及び、ＮＫ－４の添加により、ＰＩ３Ｋの下流にあるＡｋｔのリン酸化が認められることから、ＮＫ－４のこれらの作用は、ＰＩ３Ｋ－Ａｋｔカスケードの活性化によることが示唆された。 Although specific data is not shown, it has been reported that the action of nerve growth factor (NGF) on PC12 cells is inhibited by K252a, a protein kinase inhibitor highly specific to TrkA (Kase H , Et al., “Biochemical and Biophysical Research Communications”, vol. 144, pages 35-40 (1987)), and examined whether NK-4 acts on PC12-HS cells through the same pathway as NGF. , NK-4 did not inhibit the PC12-HS cell proliferation promoting action and neurite outgrowth action described below with K252a. On the other hand, the action of NK-4 inhibits the production of phosphatidylinositol (3,4,5) triphosphate by inhibiting phosphatidylinositol (3,4,5) triphosphate kinase (PI3K). LY294002 (Vlahos C. et al., “Journal of Biological Chemistry”, Vol. 269, Suppresses and ultimately suppresses the activation of serine / threonine kinase (Akt), which plays a major role in cell survival and proliferation. 5241-5248 (1994)), and the addition of NK-4 results in phosphorylation of Akt downstream of PI3K. -Suggested by activation of Akt cascade.
　さらに、ＮＫ－４は、細胞内サイクリックＡＭＰ（アデノシンモノホスフェイト）濃度の上昇誘導作用を有している。 Furthermore, NK-4 has an effect of inducing an increase in intracellular cyclic AMP (adenosine monophosphate) concentration.
　さらに、神経突起伸展誘導作用やアミロイドβペプチドにより誘導されるアポトーシスに関与することが報告されているＳＡＰＫ（Ｓｔｒｅｓｓ　ａｃｔｉｖａｔｅｄ　ｐｒｏｔｅｉｎ　ｋｉｎａｓｅ）／ＪＮＫ（ｃ－Ｊｕｎ　Ｎ－ｔｅｒｍｉｎａｌ　Ｋｉｎａｓｅ）のリン酸化（（Ｒｅｎａｅ　Ｌ．等、『Ｊｏｕｒｎａｌ　ｏｆ　Ｂｉｏｌｏｇｉｃａｌ　Ｃｈｅｍｉｓｔｒｙ』、第２７４巻、第３５４９９頁（１９９９年）、Ｗａｎｌｉ　Ｗ．等、『Ｊｏｕｒｎａｌ　ｏｆ　Ｂｉｏｌｏｇｉｃａｌ　Ｃｈｅｍｉｓｔｒｙ』、第２７７巻、第１７６４９乃至１７６５６頁（２００２年）参照）の誘導に対する作用についても、ＮＫ－４は細胞の過酸化水素障害により誘導されるＳＡＰＫ／ＪＮＫのリン酸化を抑制することから、ＮＫ－４の持つ神経突起伸展作用には、ＳＡＰＫ／ＪＮＫを介するシグナル伝達経路も関与していると判断した。 Furthermore, SAPK (Stress activated protein kinase) / JNK (c-Jun N-terminal Kinase) phosphorylation (Renae L), which has been reported to be involved in neurite outgrowth-inducing action and apoptosis induced by amyloid β peptide. "Journal of Biological Chemistry", 274, 35499 (1999), Wanli W. et al., "Journal of Biological Chemistry", 277, 17649 to 17656 (2002). Regarding the effect on induction, NK-4 suppresses phosphorylation of SAPK / JNK induced by cellular hydrogen peroxide damage. One the neurite outgrowth action, the signal transduction pathway via SAPK / JNK was also determined to be involved.
＜実験２：小脳変性運動失調症ハムスターの行動及び脳組織に及ぼすＮＫ－４投与の影響＞
　実験１で、ＮＫ－４に、神経変性抑制作用があることが確認されたので、ヒトの神経変性疾患（脊髄小脳変性症など）の好適なモデル動物として使用される小脳変性運動失調症（以下、「小脳失調症」という。）ハムスター（以下、「小脳失調症ハムスター」という）を使用して、その行動及び脳組織に及ぼすＮＫ－４投与の影響を調べた。すなわち、生後３週齢以降に小脳のプルキンエ細胞の脱落が認められ、それに引き続いて、７週齢以降に運動失調を自然発症することが知られている自然発症遺伝子突然変異（Ｎｎａ１抑制）ハムスター（Ａｋｉｔａ　Ｋ．等、『Ｊ．Ｎｅｕｒｏｇｅｎｅｔｉｃｓ』、第２１巻、第１９乃至２９頁（２００７年）参照）、株式会社林原生物化学研究所で飼育）２５匹を、表２に示す試験群１乃至５に、各群５匹を無作為に割り付けた。試験群１のハムスターには、小脳失調症発症前（３週齢）から、ＰＢＳを１０ｍｌ／ｋｇ／日投与した。試験群２乃至４のハムスターには、小脳失調症発症前（３週齢）から、ＮＫ－４を、２０μｇ／ｋｇ、１００μｇ／ｋｇ又は５００μｇ／ｋｇ／日投与した。試験群５のハムスターには、小脳失調症発症前（３週齢）から、神経細胞栄養因子として知られるインスリン様栄養因子－１（Ａｓｓａｙｐｒｏ社販売、商品名「ＩＧＦ－１、ｈｕｍａｎ」、ヒト由来、以下、「ＩＧＦ－１」と略記する。）を２５μｇ／ｋｇ／日投与した。これらの投与成分は、１日１回１０週齢まで、毎日腹腔内に投与した。小脳失調症の症状の程度と、ＮＫ－４によるその症状の改善効果を、後述するロタロッド試験、斜面耐久試験を週１回行って、ハムスターの運動協調性の改善を指標に評価した。さらに、１０週齢で、ハムスターの転倒する回数を測定後、脳を摘出し、組織学的評価を行い、併せて、血中及び脳脊髄液（ＣＳＦ）中のグルタミン酸濃度を測定した。試験群６として、試験群１乃至５で使用した小脳失調症ハムスターと同一週齢の正常ハムスター５匹に、ＰＢＳを１０ｍｌ／ｋｇ／日を、１日１回１０週齢まで、毎日腹腔内投与して、小脳失調症ハムスターと同じ試験を行った。 <Experiment 2: Effects of NK-4 administration on behavior and brain tissue of cerebellar degenerative ataxia hamster>
Since Experiment 1 confirmed that NK-4 has a neurodegenerative inhibitory effect, cerebellar degeneration ataxia (hereinafter referred to as a suitable model animal for human neurodegenerative diseases such as spinocerebellar degeneration) The hamster (hereinafter referred to as “cerebellar ataxia hamster”) was used to examine the effects of NK-4 administration on its behavior and brain tissue. In other words, detachment of Purkinje cells in the cerebellum was observed after 3 weeks of age, followed by a spontaneous gene mutation (Nna1 suppression) hamster known to spontaneously develop ataxia after 7 weeks of age ( Akita K. et al., “J. Neurogenetics”, Vol. 21, pages 19 to 29 (2007)), reared at Hayashibara Biochemical Research Institute, Inc.) 25 test groups 1 to 5 shown in Table 2 In addition, 5 animals were randomly assigned to each group. Test group 1 hamsters were administered with 10 ml / kg / day of PBS before the onset of cerebellar ataxia (3 weeks of age). The hamsters in test groups 2 to 4 were administered NK-4 at 20 μg / kg, 100 μg / kg, or 500 μg / kg / day before the onset of cerebellar ataxia (3 weeks of age). The hamsters in Test Group 5 had insulin-like trophic factor-1 known as a neuronal trophic factor-1 (sold by Assaypro, trade name “IGF-1, human”) from the beginning of cerebellar ataxia (3 weeks old) Hereinafter, abbreviated as “IGF-1”) was administered at 25 μg / kg / day. These administration components were administered intraperitoneally once daily until 10 weeks of age. The degree of cerebellar ataxia and the effect of NK-4 on improving the symptoms were evaluated by using the rotarod test and slope endurance test described later once a week, with the improvement of hamster motor coordination as an index. Furthermore, at the age of 10 weeks, after measuring the number of hamster falls, the brain was removed and histologically evaluated, and the glutamic acid concentration in blood and cerebrospinal fluid (CSF) was also measured. As test group 6, 5 normal hamsters of the same age as the cerebellar ataxia hamsters used in test groups 1 to 5 were subjected to intraperitoneal administration of 10 ml / kg / day of PBS once daily until 10 weeks of age. Then, the same test as the cerebellar ataxia hamster was conducted.
＜ロタロッド試験＞
　ハムスターが、ロタロッドの回転にあわせて歩行運動し、ロタロッド上に留まるために行う運動の継続時間を運動協調性の指標とし使用した。すなわち、ハムスターを一定速度（６ｒｐｍ）で回転するロタロッド装置（株式会社林原生物化学研究所製造、ロタロッドの直径６０ｍｍ）に乗せ、ロタロッドから落下するまでの時間を測定した（Ｆｅｒｎａｎｄｅｚ等、『Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．ＵＳＡ』、第９５号、第１２５３乃至１２５８頁（１９９８年）参照）。試験は、１匹のハムスターについて６回行い、最初から５回は、回転運動に馴化させるための予備運動試験とし、６回目の試験で、ロタロッドから落下するまでの時間（以下、「落下時間」という）を計測して、各群５匹の平均を求めた。結果を表２に示す。なお、落下時間は１８０秒まで計測した。さらに、この結果に基づき、小脳失調症ハムスターにＰＢＳを投与した場合（試験群１）の週齢と落下時間の関係を示すグラフを作成し、小脳失調症ハムスターにＮＫ－４（試験群２乃至４）又はＩＧＦ－１（試験群５）を投与した時の、５週齢以降（ＮＫ－４又はＩＧＦ－１投与２週間以降）について、各試験群の各週齢のハムスターの落下時間が、試験群１のハムスターの何週齢の落下時間と同じになるかをグラフから求め、試験群２乃至５のハムスターの小脳失調症の進行が遅延された日数を求めた（＝試験群２乃至５の週齢×７（日）－計算から求めた試験群２乃至５の週齢と同じ落下時間を示す試験群１の週齢×７（日））。結果を表３に示す。なお、表３に示すように、正常ハムスターでは、試験した何れの週齢においても、１８０秒間では、ロッドから落下したハムスターは認められなかったので、１８０秒以内に落下した場合には、ハムスターが小脳失調症を発症して運動協調性が低下したと判断した。 <Rotarod test>
The hamster walked with the rotation of the rotarod and used the duration of the exercise to stay on the rotarod as an indicator of motor coordination. That is, a hamster was placed on a rotarod apparatus (manufactured by Hayashibara Biochemical Laboratories Co., Ltd., rotarod diameter 60 mm) rotating at a constant speed (6 rpm), and the time until it dropped from the rotarod was measured (Fernandez et al., “Proc. Natl. Acad. Sci. USA, 95, 1253-1258 (1998)). The test is conducted 6 times for one hamster, and the first 5 times is a preliminary motion test for acclimatization to the rotational motion. In the sixth test, the time until it falls from the rotarod (hereinafter referred to as “fall time”) And the average of 5 animals in each group was obtained. The results are shown in Table 2. The falling time was measured up to 180 seconds. Further, based on this result, a graph showing the relationship between the age of the cerebellar ataxia hamster when PBS was administered (test group 1) and the fall time was prepared. 4) or after 5 weeks of age (after 2 weeks of NK-4 or IGF-1 administration) when IGF-1 (test group 5) was administered, the fall time of each weekly hamster in each test group was tested From the graph, the number of weeks of fall of the hamsters in group 1 was the same as the fall time, and the number of days in which progression of cerebellar ataxia in hamsters in test groups 2 to 5 was delayed was calculated (= in test groups 2 to 5). Week age x 7 (days)-Test group 1 week age x 7 (days) showing the same drop time as that of test groups 2-5 as determined from the calculation. The results are shown in Table 3. As shown in Table 3, in any normal hamster, no hamster dropped from the rod was observed in 180 seconds at any age tested. It was judged that cerebellar ataxia developed and motor coordination decreased.
＜斜面耐久試験＞
　ハムスターの頭部を上にして、傾斜角度の変えられる板の上に乗せ、５秒間静止できる角度を判定して、斜面耐久傾斜角度（Ｒｉｖｌｉｎ等、『Ｊ．Ｎｅｕｒｏｓｕｒｇ．』、第４７巻、第５７７－５８１頁（１９９７年）参照）とした。傾斜角度は２５度から開始し、５度ずつ上昇させた。静止が５秒未満で落下した場合には、その傾斜角度から、１度間隔で角度を減じて、５秒間静止できる角度を判定して、斜面耐久傾斜角度を測定して、各群５匹の平均を求めた。結果を表４に示す。 <Slope durability test>
With the head of the hamster facing up, place it on a plate that can change the tilt angle and determine the angle at which it can stand for 5 seconds. 577-581 (1997)). The tilt angle started from 25 degrees and increased by 5 degrees. If the vehicle falls in less than 5 seconds, subtract the angle at 1 degree intervals from the tilt angle, determine the angle at which it can stand for 5 seconds, measure the slope endurance tilt angle, The average was calculated. The results are shown in Table 4.
＜ハムスターの転倒する回数＞
　１０週齢のハムスターを１匹ずつ飼育ケージに入れ、１分間に転倒する回数を肉眼観察により計測して、各群５匹の平均を求めた。結果を表５に示す。なお、この試験で使用した小脳失調症ハムスターは、９週齢以降に転倒頻度が増加することが知られている。 <Number of hamster falls>
One 10-week-old hamster was put in a breeding cage one by one, and the number of falls per minute was measured by visual observation, and the average of 5 animals in each group was determined. The results are shown in Table 5. In addition, it is known that the cerebellar ataxia hamster used in this test increases the frequency of falls after 9 weeks of age.
＜組織学的評価＞
　転倒回数を確認などの運動協調性に関する試験終了後の１０週齢ハムスターにペントバルビタールを５０ｍｇ／ｋｇ腹腔内投与し、麻酔下で、後大静脈より放血、致死せしめ、大脳及び小脳を摘出し、１０容積％ホルマリン溶液で固定後、上部より大脳及び小脳を、デジタルカメラで、一定の高さから撮影し、それぞれの矢状方向及び水平方向の直径を計測した。矢状方向長２×水平方向長×０．５を計算して、それぞれ大脳体積及び小脳体積とし、各群５匹の平均を求めた。結果を表５に示す。さらに、この試験で使用した小脳失調症ハムスターは、抑制性神経細胞のプルキンエ細胞に加えて、興奮性神経細胞である顆粒細胞も細胞密度が減少することが知られている。そこで、矢状方向に切り出した小脳切片を、常法によりヘマトキシリン・エオシン染色し、検鏡してプルキンエ細胞層（小葉Ｉ乃至Ｘ）にある全プルキンエ細胞数及び単位面積当たりの顆粒細胞数を計測すると共に、小脳白質に脱髄が認められる個体数を確認した。結果を表５に併せて示す。なお、大脳体積は、各試験群間で有意の差が認められなかったので、表５には小脳体積の計算結果のみを示す。 <Histological evaluation>
50 mg / kg of pentobarbital was intraperitoneally administered to a 10-week-old hamster after completion of the test for motor coordination such as confirming the number of falls After fixing with a 10% by volume formalin solution, the cerebrum and cerebellum were photographed from above at a certain height with a digital camera, and the respective sagittal and horizontal diameters were measured. Sagittal length 2 × horizontal length × 0.5 was calculated as cerebrum volume and cerebellum volume, respectively, and the average of 5 animals in each group was determined. The results are shown in Table 5. Furthermore, the cerebellar ataxia hamster used in this test is known to have a reduced cell density in addition to Purkinje cells, which are inhibitory neurons, and granule cells, which are excitatory neurons. Therefore, cerebellar slices cut in the sagittal direction were stained with hematoxylin and eosin by a conventional method, and microscopically measured to determine the total number of Purkinje cells in the Purkinje cell layer (lobules I to X) and the number of granule cells per unit area. In addition, the number of individuals with demyelination in the cerebellar white matter was confirmed. The results are also shown in Table 5. In addition, since the cerebral volume did not recognize a significant difference between each test group, Table 5 shows only the calculation result of the cerebellum volume.
＜血中及び脳脊髄液（ＣＳＦ）中のグルタミン酸濃度＞
　哺乳類の中枢神経において記憶・学習などの高次機能を調節する主要な興奮性神経伝達化合物であり、抑制性神経伝達化合物であるγ－アミノ酪酸（ＧＡＢＡ）の合成に使用されるグルタミン酸の量を測定した。すなわち、上記ハムスターの脳摘出の際に、後大静脈より採血した後、ＣＳＦを採取して、後述のグルタミン酸濃度の測定に供した。血中及びＣＳＦ中のグルタミン酸測定には、グルタミン酸測定キット（Ｉｎｖｉｔｒｏｇｅｎ社販売、商品名「ＡｍｐｌｅｘＴＭ　Ｒｅｄ　Ｇｌｕｔａｍｉｃ　Ａｃｉｄ／Ｇｌｕｔａｍａｔｅ　Ｏｘｉｄａｓｅ　Ａｓｓａｙ　Ｋｉｔ」）を使用した。結果を表５に併せて示す。なお、血中のグルタミン酸量は、各試験群間で有意の差が認められなかったため、表５にはＣＳＦの測定結果のみを示す。 <Glutamate concentration in blood and cerebrospinal fluid (CSF)>
The amount of glutamate used in the synthesis of γ-aminobutyric acid (GABA), a major excitatory neurotransmitter that regulates higher-order functions such as memory and learning in the mammalian central nervous system. It was measured. That is, at the time of brain extraction of the hamster, blood was collected from the posterior vena cava, then CSF was collected and used for measurement of glutamic acid concentration described later. For measurement of glutamic acid in blood and CSF, a glutamic acid measurement kit (trade name “Amplex ™ Red Acid Acid / Glutamate Oxidase Assay Kit”, sold by Invitrogen) was used. The results are also shown in Table 5. Since no significant difference was observed between the test groups in the amount of glutamic acid in blood, Table 5 shows only the CSF measurement results.
Figure JPOXMLDOC01-appb-T000025
Figure JPOXMLDOC01-appb-T000025
Figure JPOXMLDOC01-appb-T000026
Figure JPOXMLDOC01-appb-T000026
Figure JPOXMLDOC01-appb-T000027
Figure JPOXMLDOC01-appb-T000027
Figure JPOXMLDOC01-appb-T000028
Figure JPOXMLDOC01-appb-T000028
　表２から明らかなように、４週齢の正常ハムスター（試験群６）の落下時間は１８０秒以上であったのに対して、同じ４週齢の小脳失調症ハムスターを使用した試験群１（ＰＢＳ投与群）の落下時間は１０８±１０秒で、すでに、正常ハムスターに比較して有意な落下時間の短縮が認められ、以後、加齢と共に、さらに短縮した。１０週齢では、ロタロッドに留まることはできず、すぐに落下（０秒）した。これに対して、ＮＫ－４を投与したハムスターでは、投与開始後１週目（４週齢）から、用量依存的に、落下時間短縮の抑制が認められ、試験群３（１００μｇ／ｋｇ・体重）、及び、試験群４（５００μｇ／ｋｇ・体重）では、試験群１に比較して有意な落下時間短縮の抑制効果が認められた。この抑制効果は、試験終了の１０週齢（投与７週間）まで持続した。試験群２（２０μｇ／ｋｇ・体重）でもＮＫ－４投与開始後２週目（５週齢）以降に、試験群１よりも落下時間の短縮の抑制効果は認められたが、試験群３及び試験群４に比較してその効果は弱かった。また、運動神経変性疾患の治療に効果があるとされているＩＧＦ－１を投与した場合（試験群５）には、落下時間短縮の抑制効果はほとんど認められなかった。同様に、落下時間短縮の遅延日数を示す表３からも明らかなように、１０週齢（ＮＫ－４投与期間４９日）時点でも、試験群２乃至４では、各々、３２、３６、及び、３５日の、落下時間短縮の遅延が認められた。この試験期間を通して、試験群３及び４で最も高い落下時間短縮の抑制効果が認められ、小脳失調症の発症遅延効果が認められた。しかし、ＩＧＦ－１を投与した試験群５ではほとんど症状発症遅延効果は認められず、１０週齢時点では、落下時間に試験群１との差は認められなかった。この結果は、ＮＫ－４が、ヒトの神経変性症及びその病態や症状の治療剤として利用できることを物語っている。 As is clear from Table 2, the drop time of a 4-week-old normal hamster (Test Group 6) was 180 seconds or more, whereas Test Group 1 (using the same 4-week-old cerebellar ataxia hamster) ( The fall time of the PBS administration group was 108 ± 10 seconds, and a significant shortening of the fall time was already observed as compared with normal hamsters. At the age of 10 weeks, it was not possible to stay on the rotarod and it immediately dropped (0 seconds). In contrast, in the hamsters administered with NK-4, the reduction of the fall time was observed in a dose-dependent manner from the first week (4 weeks of age) after the start of administration, and in test group 3 (100 μg / kg / body weight). ) And test group 4 (500 μg / kg · body weight), a significant effect of reducing the drop time was observed compared to test group 1. This inhibitory effect lasted until the end of the study at 10 weeks of age (7 weeks of administration). Even in Test Group 2 (20 μg / kg / body weight), after 2 weeks (5 weeks old) after the start of NK-4 administration, the effect of reducing the drop time was observed more than in Test Group 1, but Test Group 3 and Compared with test group 4, the effect was weak. In addition, when IGF-1 which is considered to be effective in the treatment of motor neurodegenerative diseases was administered (Test Group 5), the effect of suppressing the fall time was hardly observed. Similarly, as is apparent from Table 3 showing the number of days for delaying the fall time reduction, even at 10 weeks of age (NK-4 administration period 49 days), in test groups 2 to 4, 32, 36, and A delay in shortening the fall time was observed on the 35th. Throughout this test period, the highest effect of reducing the fall time was observed in Test Groups 3 and 4, and the onset delay effect of cerebellar ataxia was observed. However, almost no symptom onset delay effect was observed in Test Group 5 administered with IGF-1, and no difference in drop time from Test Group 1 was observed at 10 weeks of age. This result demonstrates that NK-4 can be used as a therapeutic agent for human neurodegeneration and its pathological conditions and symptoms.
　表４の結果から明らかなように、試験群６（正常ハムスター）の斜面耐久傾斜角度は、試験期間を通して４６度から５２度の範囲内であったが、小脳失調症ハムスターを使用した試験群１（ＰＢＳ投与群）では、試験開始時には、すでに、４０．６±０．３度と有意に低く、加齢に伴い、減少して、８週齢以降はさらに低下し、１０週齢では３５．８±１．０度となった。試験群５（ＩＧＦ－１投与）では、４週齢で４４．４±０．２度となって、試験群１の４０．６±０．３度に比較して有意な斜面耐久傾斜角度の低下抑制効果が認められたものの、それ以降は試験群１と同様に斜面耐久傾斜角度が低下し、５週齢以降は有意な改善は認められなかった。これに対して、試験群２乃至４（ＮＫ－４投与）では、試験に使用した何れの投与量でも、試験期間を通して耐久傾斜角度低下は認められず、高い斜面耐久傾斜角度低下抑制効果が認められた。１０週齢での試験群２乃至４（ＮＫ－４投与）の斜面耐久傾斜角度は、各々、４５．８±０．６、５１．６±０．６、５１．８±０．４度となり、いずれも試験群１（ＰＢＳ投与）や試験群５（ＩＧＦ－１投与）に比較して有意に高かった。 As is apparent from the results in Table 4, the slope endurance slope angle of test group 6 (normal hamster) was in the range of 46 to 52 degrees throughout the test period, but test group 1 using cerebellar ataxia hamsters. In the PBS administration group, it was already significantly low at 40.6 ± 0.3 degrees at the start of the test, decreased with aging, further decreased after 8 weeks of age, and 35. It was 8 ± 1.0 degrees. In test group 5 (IGF-1 administration), 44.4 ± 0.2 degrees at 4 weeks of age, which is a significant slope durability inclination angle compared to 40.6 ± 0.3 degrees in test group 1 Although a decrease suppressing effect was observed, thereafter, the slope durability inclination angle decreased as in Test Group 1, and no significant improvement was observed after 5 weeks of age. In contrast, in test groups 2 to 4 (NK-4 administration), no decrease in endurance tilt angle was observed throughout the test period at any dose used in the test, and a high slope endurance tilt angle decrease suppression effect was observed. It was. The slope endurance slopes of the test groups 2 to 4 (NK-4 administration) at 10 weeks of age are 45.8 ± 0.6, 51.6 ± 0.6, and 51.8 ± 0.4 degrees, respectively. Both were significantly higher than those in Test Group 1 (PBS administration) and Test Group 5 (IGF-1 administration).
　表５の結果から明らかなように、１０週齢の正常ハムスター（試験群６）では、転倒は認められないのに対して、試験群１（ＰＢＳ投与）では、１２．８±０．５回／分の転倒が認められた。これに対して、試験群５（ＩＧＦ－１投与）では、１１．４±０．４回／分と試験群１と比較して、やや減少傾向にあったが、有意な転倒減少効果は認められなかった。これに対して、試験群２乃至４（ＮＫ－４投与）では、各々、４．０±１．０、１．６±０．９、１．２±０．８回／分と、いずれの群でも有意な転倒回数の減少が認められた。また、試験群１（ＰＢＳ投与）の小脳体積は、１０週齢で６４．６±９．４ｍｍ３と、同一週齢の正常ハムスター（試験群６）の９４．７±１１．４ｍｍ３に比較して有意な萎縮が認められた。これに対し、試験群５（ＩＧＦ－１投与）では、７７．６±６．１（ｍｍ３）と、試験群１に比して有意な小脳の萎縮抑制効果が認められた。また、ＮＫ－４投与群でも用量依存的な小脳萎縮抑制効果が認められ、２０、１００、５００μｇ／ｋｇ投与群で、それぞれ、７６．０±８．２、７７．０±２．８、８０．５±１０．８ｍｍ３となった（全てＰ＜０．０５で有意）。一方、何れの群においても大脳体積に有意の差は認められなかった（データ未掲載）。以上の結果から、ＮＫ－４は、小脳失調症ハムスターの運動協調性を改善するとともに、小脳萎縮を効果的に抑制すること、及び、その効果はＩＧＦ－１よりも優れていることが判明した。 As is apparent from the results in Table 5, no fall was observed in the 10-week-old normal hamster (test group 6), whereas in test group 1 (PBS administration), 12.8 ± 0.5 times A fall of / min was observed. In contrast, in test group 5 (IGF-1 administration), 11.4 ± 0.4 times / min was slightly decreased compared to test group 1, but a significant fall-reducing effect was observed. I couldn't. In contrast, in test groups 2 to 4 (NK-4 administration), 4.0 ± 1.0, 1.6 ± 0.9, and 1.2 ± 0.8 times / min, respectively, There was also a significant decrease in the number of falls in the group. In addition, the cerebellar volume of test group 1 (PBS administration) was 64.6 ± 9.4 mm 3 at 10 weeks of age and 94.7 ± 11.4 mm 3 of normal hamsters (test group 6) of the same age. As a result, significant atrophy was observed. In contrast, in test group 5 (IGF-1 administration), a significant cerebellar atrophy suppression effect was observed as compared to test group 1, which was 77.6 ± 6.1 (mm 3 ). In addition, a dose-dependent cerebellar atrophy suppression effect was also observed in the NK-4 administration group, and 76.0 ± 8.2, 77.0 ± 2.8, 80 in the 20, 100, and 500 μg / kg administration groups, respectively. 5 ± 10.8 mm 3 (all significant at P <0.05). On the other hand, there was no significant difference in cerebral volume in any group (data not shown). From the above results, it was found that NK-4 improves motor coordination of cerebellar ataxia hamsters, effectively suppresses cerebellar atrophy, and its effect is superior to IGF-1. .
　また、表５の結果から明らかなように、小脳皮質の顆粒細胞層にある顆粒細胞密度は、１０週齢の正常ハムスター（試験群６）が、４８０±６個／２０，０００μｍ２であるのに対し、小脳失調症ハムスターにＰＢＳを投与した場合（試験群１）は３８０±４個／２０，０００μｍ２にまで、有意に減少した。試験群５（ＩＧＦ－１投与）の顆粒細胞密度は、３７１±１１個／２００００μｍ２と、試験群１と差は認められなかった。試験群２乃至４（ＮＫ－４投与）では、各々、４０８±８、４１９±６、４３６±７個／２０，０００μｍ２となり、ＮＫ－４の投与量に依存した顆粒細胞密度減少抑制効果が認められた。また、具体的なデータは示さないが、小脳実質組織の顕微鏡観察では、試験群１や５では顆粒細胞の萎縮や変性が顕著であったのに対して、試験群２乃至４では、顆粒細胞の萎縮や変性が抑制されていることが確認された。さらに、小脳失調症ハムスターにＰＢＳを投与した場合（試験群１）では、小脳白質の脱髄が全個体（５匹中５匹）に認められたが、ＮＫ－４投与群では試験群２乃至４（ＮＫ－４投与）では、各々、５匹中４匹、５匹中１匹、５匹中０匹に脱髄が認められたのみあり、これらのＮＫ－４投与群では、プルキンエ細胞の持つ樹状突起の萎縮の抑制も確認された。この結果は、ＮＫ－４が、小脳白質の脱髄の抑制、則ち、プルキンエ細胞の変性や脱落の抑制剤としても有用であり、プルキンエ細胞をはじめとする神経細胞の細胞突起の萎縮抑制剤乃至伸展促進剤としても利用できることを物語っている。 As is clear from the results in Table 5, the granule cell density in the granule cell layer of the cerebellar cortex is 480 ± 6 cells / 20,000 μm 2 for 10-week-old normal hamsters (test group 6). On the other hand, when PBS was administered to cerebellar ataxia hamsters (Test Group 1), the number decreased significantly to 380 ± 4 / 20,000 μm 2 . The granule cell density in test group 5 (IGF-1 administration) was 371 ± 11 cells / 20,000 μm 2, which was not different from test group 1. In test groups 2 to 4 (NK-4 administration), 408 ± 8, 419 ± 6, 436 ± 7 cells / 20,000 μm 2 were obtained, respectively. Admitted. In addition, although specific data are not shown, in microscopic observation of the cerebellar parenchyma, in the test groups 1 and 5, granule cell atrophy and degeneration were significant, whereas in the test groups 2 to 4, granule cells were observed. It was confirmed that atrophy and degeneration were suppressed. Furthermore, when PBS was administered to a cerebellar ataxia hamster (test group 1), demyelination of cerebellar white matter was observed in all individuals (5 of 5 animals), whereas in the NK-4 administration group, test groups 2 to 4 (NK-4 administration), demyelination was only observed in 4 out of 5 animals, 1 out of 5 animals, and 0 out of 5 animals. In these NK-4 administration groups, Purkinje cell Suppression of dendritic atrophy was also confirmed. This result shows that NK-4 is also useful as an inhibitor of cerebellar white matter demyelination, ie, an inhibitor of Purkinje cell degeneration and loss, and an atrophy inhibitor of neuronal cell processes such as Purkinje cells. It also tells us that it can be used as an extension accelerator.
　さらに、表５の結果から明らかなよう、ＣＳＦ（脊髄液）中のグルタミン酸濃度は、ＮＫ－４の投与量に依存して回復し（試験群２乃至４）、試験群４（ＮＫ－４を５００μｇ／ｋｇ投与）では、試験群１（ＰＢＳ投与）に比較して、有意に上昇した。 Furthermore, as is clear from the results in Table 5, the glutamic acid concentration in CSF (spinal fluid) recovered depending on the dose of NK-4 (test groups 2 to 4), and test group 4 (NK-4) In the case of 500 μg / kg administration), the increase was significantly higher than that in test group 1 (PBS administration).
　以上のように、ＮＫ－４を投与した小脳失調症ハムスターでは、ロタロッド試験、斜面耐久試験、転倒回数の全ての試験項目で症状改善が認められ、その効果は、ＮＫ－４の投与量に依存し、投与量が１００及び５００μｇ／ｋｇ・体重／日で、特に高い効果が得られた。この結果は、腹腔内に投与したＮＫ－４は、脳神経細胞に作用し、小脳失調症を抑制したことを示している。また、ＮＫ－４の投与により小脳失調ハムスターのＣＳＦ中のグルタミン酸量の低下が抑制されたことから、ＮＫ－４は神経細胞の活性化を介して、神経変性に伴う神経細胞の機能低下を抑制し、運動能や学習能の低下を抑制できることを物語っている。また、これらの結果は、ＮＫ－４が、ヒトの神経変性症やそれに伴う種々の病態や臨床症状に対する全身投与可能な治療剤として有用であることを物語っている。なお、小脳失調ハムスターの体重を試験終了（１０週齢）まで毎週１回計測して、各群の平均を求めたところ、いずれの週齢においても、ＰＢＳ、ＮＫ－４、ＩＧＦ－１投与群間に有意な差は認められなかったことから、ＮＫ－４は生体へ長期間連続投与しても安全性が高い化合物と判断した。 As described above, in cerebellar ataxia hamsters administered with NK-4, symptom improvement was observed in all test items of rotarod test, slope endurance test, and number of falls, and the effect depends on the dose of NK-4 However, particularly high effects were obtained at doses of 100 and 500 μg / kg · body weight / day. This result indicates that NK-4 administered intraperitoneally acted on cerebral neurons and suppressed cerebellar ataxia. In addition, NK-4 suppressed the decrease in glutamate content in CSF of cerebellar ataxic hamsters, and NK-4 suppressed the decrease in neuronal function associated with neurodegeneration through the activation of neurons. And it shows that it can control the decline of motor ability and learning ability. These results also indicate that NK-4 is useful as a systemically administrable agent for human neurodegeneration and various pathological and clinical symptoms associated therewith. The body weight of the cerebellar ataxia hamster was measured once a week until the end of the test (10 weeks of age) and the average of each group was determined. At any age, PBS, NK-4, IGF-1 administration group Since no significant difference was observed, NK-4 was judged to be a highly safe compound even when continuously administered to a living body for a long period of time.
＜実験３：ＮＫ－４以外の色素化合物の作用＞
　実験１及び２において、ＮＫ－４に細胞傷害性因子に対する保護作用、神経変性抑制作用、小脳失調症を引き起こすプルキンエ細胞減少抑制、神経細胞減少抑制作用などがあることが確認されたので、ＮＫ－４以外の色素化合物（以下、単に「化合物」という場合がある。）についても、同様の作用があるかどうかを検討した。すなわち、表６に示す化学式２、４乃至９で表される化合物に加えて、下記化学式１０乃至２４１で表される２３２種類（合計２３９種類）の化合物について、以下に示す細胞増殖促進活性（評価法Ａ）及び神経突起伸展率（評価法Ｂ）により、ＰＣ１２－ＨＳ細胞に対する細胞増殖活性と神経突起伸展促進作用の有無を調べた結果を表６に併せて示す。なお、下記試験において、各々の判定基準よりも低い効果しか認められなかった場合、表６では空欄とした。また、本明細書では、化学式２、４乃至２４１で表される化合物は、それぞれ、表６に示す略号（ＮＫ番号）で表記する場合がある。 <Experiment 3: Action of dye compounds other than NK-4>
In Experiments 1 and 2, it was confirmed that NK-4 has a protective action against cytotoxic factors, an inhibitory effect on neurodegeneration, an inhibitory effect on Purkinje cell reduction that causes cerebellar ataxia, and an inhibitory action on neuronal cell loss. It was examined whether or not a dye compound other than 4 (hereinafter sometimes simply referred to as “compound”) has the same effect. That is, in addition to the compounds represented by the chemical formulas 2, 4 to 9 shown in Table 6, 232 types (total 239 types) of compounds represented by the following chemical formulas 10 to 241 have the following cell growth promoting activity (evaluation) Table 6 also shows the results of examining the presence of cell proliferation activity and neurite outgrowth promoting action on PC12-HS cells by method A) and neurite outgrowth rate (evaluation method B). In addition, in the following test, when only an effect lower than each criterion was recognized, it was left blank in Table 6. In this specification, the compounds represented by Chemical Formulas 2, 4 to 241 may be represented by abbreviations (NK numbers) shown in Table 6, respectively.
Figure JPOXMLDOC01-appb-C000029
Figure JPOXMLDOC01-appb-C000029
Figure JPOXMLDOC01-appb-C000030
Figure JPOXMLDOC01-appb-C000030
Figure JPOXMLDOC01-appb-C000031
Figure JPOXMLDOC01-appb-C000031
Figure JPOXMLDOC01-appb-C000032
Figure JPOXMLDOC01-appb-C000032
Figure JPOXMLDOC01-appb-C000033
Figure JPOXMLDOC01-appb-C000033
Figure JPOXMLDOC01-appb-C000034
Figure JPOXMLDOC01-appb-C000034
Figure JPOXMLDOC01-appb-C000035
Figure JPOXMLDOC01-appb-C000035
Figure JPOXMLDOC01-appb-C000036
Figure JPOXMLDOC01-appb-C000036
Figure JPOXMLDOC01-appb-C000037
Figure JPOXMLDOC01-appb-C000037
Figure JPOXMLDOC01-appb-C000038
Figure JPOXMLDOC01-appb-C000038
Figure JPOXMLDOC01-appb-C000039
Figure JPOXMLDOC01-appb-C000039
Figure JPOXMLDOC01-appb-C000040
Figure JPOXMLDOC01-appb-C000040
Figure JPOXMLDOC01-appb-C000041
Figure JPOXMLDOC01-appb-C000041
Figure JPOXMLDOC01-appb-C000042
Figure JPOXMLDOC01-appb-C000042
Figure JPOXMLDOC01-appb-C000043
Figure JPOXMLDOC01-appb-C000043
Figure JPOXMLDOC01-appb-C000044
Figure JPOXMLDOC01-appb-C000044
Figure JPOXMLDOC01-appb-C000045
Figure JPOXMLDOC01-appb-C000045
Figure JPOXMLDOC01-appb-C000046
Figure JPOXMLDOC01-appb-C000046
Figure JPOXMLDOC01-appb-C000047
Figure JPOXMLDOC01-appb-C000047
Figure JPOXMLDOC01-appb-C000048
Figure JPOXMLDOC01-appb-C000048
Figure JPOXMLDOC01-appb-C000049
Figure JPOXMLDOC01-appb-C000049
Figure JPOXMLDOC01-appb-C000050
Figure JPOXMLDOC01-appb-C000050
Figure JPOXMLDOC01-appb-C000051
Figure JPOXMLDOC01-appb-C000051
＜試験試料＞
　表６に示す２３９種類の化合物は、水に難溶性のものが多いため、ＮＫ－４の場合と同様に、ＤＭＳＯ（ＳＩＧＭＡ社販売、カタログ番号「Ｄ８４１８」）に５ｍｇ／ｍｌの濃度で溶解した後、Ｍｉｌｌｅｘ－ＬＧ（Ｍｉｌｌｉｐｏｒｅ社販売、製品番号「ＬＬＧ０２５ＳＳ」、ＤＭＳＯ耐性）で膜ろ過し、遮光して２５℃で保存した。使用時には、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地（日水製薬）で２００倍以上に希釈して試験試料を調製し、試験に供した。これらの化合物は、いずれも株式会社林原生物化学研究所で合成したものを使用した。 <Test sample>
Since 239 types of compounds shown in Table 6 are hardly soluble in water, they were dissolved in DMSO (SIGMA, catalog number “D8418”) at a concentration of 5 mg / ml, as in the case of NK-4. Thereafter, the membrane was filtered with Millex-LG (sold by Millipore, product number “LLG025SS”, DMSO resistant), and stored at 25 ° C. protected from light. At the time of use, a test sample was prepared by diluting 200-fold or more with 10% by volume FBS-added D-MEM medium (Nissui Pharmaceutical), and subjected to the test. These compounds were all synthesized by Hayashibara Biochemical Laboratories.
＜評価法Ａ：神経細胞増殖促進作用の評価法＞
　実験１と同じ方法で、ＰＣ１２－ＨＳ細胞を、予めコラーゲンコートした９６ウエルマイクロプレートに５×１０３個／ウエルになるように１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈し、１００μｌ／ウエルで播種した。２４時間後に１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈し、１００ｎｇ／ｍｌに調整した各試験試料を１００μｌ／ウエル添加し、３７℃、５容積％ＣＯ２インキュベーター内で３日間培養した。３日目に培養上清を除去し、１０質量％Ａｌａｍａｒ　ｂｌｕｅ（Ｔｒｅｋ　Ｄｉａｇｎｏｓｔｉｃ社販売）／１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地を２００μｌ／ウエルずつ添加し、６時間、３７℃、５容積％ＣＯ２インキュベーター中で培養し、蛍光プレートリーダー（日本モレキュラーディバイス社販売）で５４４－５９０ｎｍの蛍光強度を測定した。各試験サンプルの細胞増殖促進作用は、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地を１００μｌ／ウエル添加した対照の値を１００とした時の相対値が１４０乃至１９９の場合をＮＫ－４と同等（○）、２００以上をＮＫ－４より強い作用（◎）と判定した。結果を表６に併せて示す。 <Evaluation method A: Evaluation method of nerve cell proliferation promoting action>
In the same manner as in Experiment 1, PC12-HS cells were diluted with D-MEM medium supplemented with 10% by volume FBS to a pre-collagen-coated 96-well microplate at 5 × 10 3 cells / well, and 100 μl / well. Sowing. After 24 hours, each test sample diluted with 10 volume% FBS-added D-MEM medium and adjusted to 100 ng / ml was added at 100 μl / well, and cultured in a 37 ° C., 5 volume% CO 2 incubator for 3 days. On the third day, the culture supernatant was removed, and 10% by mass Alamar blue (available from Trek Diagnostics) / 10 volume% FBS-added D-MEM medium was added at 200 μl / well for 6 hours at 37 ° C., 5 volume% CO. The cells were cultured in two incubators, and the fluorescence intensity at 544-590 nm was measured with a fluorescence plate reader (sold by Nippon Molecular Devices). The cell growth promoting effect of each test sample is equivalent to that of NK-4 when the relative value is 140 to 199 when the control value obtained by adding 100 μl / well of 10% FBS-added D-MEM medium is 100 (○ ), 200 or more were judged to be stronger (◎) than NK-4. The results are also shown in Table 6.
＜評価法Ｂ：神経突起伸展作用の評価法＞
　細胞増殖促進活性の測定の場合と同様に、ＰＣ１２－ＨＳ細胞を、予めコラーゲンコートした９６ウエルマイクロプレートに５×１０３個／ウエルになるように１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈し、１００μｌ／ウエルで播種した。２４時間後に、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈して４００ｎｇ／ｍｌに調整した各試験試料５０μｌ／ウエルと、２０ｎｇ／ｍｌＮＧＦ（Ｃｈｅｍｉｃｏｎ社販売、マウス由来、終濃度５ｎｇ／ｍｌ）含有１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地５０μｌ／ウエルとを加えて、３日間培養した。培養３日目に１０容積％グルタルアルデヒドで室温２０分間固定した。対照として１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地のみで３日間培養したＰＣ１２－ＨＳ細胞をグルタルアルデヒドで固定した。固定した細胞を、顕微鏡下で観察し、神経突起伸展の有無を評価し、神経突起伸展率が３０％以上の場合をＮＫ－４と同等以上（○）と判定した。なお、神経突起伸展率（％）は、顕微鏡下で、一視野に約１００個の細胞を含む倍率で細胞を観察し、細胞体の２倍以上の神経突起を有する細胞数をカウントし、同一視野内にある全細胞数で除し、１００倍して求めた。また、この実験系にＮＧＦのみを添加（５ｎｇ／ｍｌ）したときの神経突起伸展率は５％程度であった。結果を表６に併せて示す。 <Evaluation method B: Evaluation method of neurite extension action>
As in the case of the measurement of cell growth promoting activity, PC12-HS cells were diluted with D-MEM medium supplemented with 10% by volume FBS to a 96-well microplate previously coated with collagen at 5 × 10 3 cells / well. And 100 μl / well. 24 hours later, each test sample was diluted with 10% by volume FBS-added D-MEM medium and adjusted to 400 ng / ml, 50 μl / well, and 20 ng / ml NGF (available from Chemicon, mouse origin, final concentration 5 ng / ml) 10 50% l / well of D-MEM medium supplemented with volume% FBS was added and cultured for 3 days. On the third day of culture, the cells were fixed with 10% by volume glutaraldehyde for 20 minutes at room temperature. As a control, PC12-HS cells cultured for 3 days only in 10% by volume FBS-added D-MEM medium were fixed with glutaraldehyde. The fixed cells were observed under a microscope to evaluate the presence or absence of neurite outgrowth, and when the neurite outgrowth rate was 30% or more, it was determined to be equal to or better than NK-4 (◯). The neurite outgrowth rate (%) is the same by observing cells under a microscope at a magnification including about 100 cells in one field, counting the number of cells having neurites more than twice the cell body, Dividing by the total number of cells in the field of view and multiplying by 100 was obtained. Further, when only NGF was added to this experimental system (5 ng / ml), the neurite extension rate was about 5%. The results are also shown in Table 6.
＜評価法Ｃ：アミロイドβフラグメントによる細胞傷害に対する抑制作用＞
　評価法Ａで細胞増殖促進作用の認められた試験試料について、実験１と同じ方法で、アミロイドβフラグメントによる細胞傷害に対する細胞保護効果があるかどうかを検討した。試験試料無添加の場合に比して、アミロイドβフラグメントによる細胞傷害を有意に抑制した場合に抑制効果ありとして○を付した。結果を表６に併せて示す。 <Evaluation Method C: Inhibitory Action on Cell Damage by Amyloid β Fragment>
The test sample in which the cell growth promoting action was recognized by the evaluation method A was examined by the same method as in Experiment 1 to determine whether it had a cytoprotective effect against cell damage caused by amyloid β fragment. As compared with the case where the test sample was not added, when the cell damage by the amyloid β fragment was significantly suppressed, it was marked as having an inhibitory effect. The results are also shown in Table 6.
Figure JPOXMLDOC01-appb-T000052
Figure JPOXMLDOC01-appb-T000052
　表６の結果に示すように、試験した２３９種の化合物は、細胞増殖促進作用（評価法Ａ）や神経突起伸展促進作用（評価法Ｂ）があることが判明した。とりわけ、ＮＫ－１９（化学式４で表される化合物）、ＮＫ－５３（化学式５で表される化合物）、ＮＫ－１００（化学式６で表される化合物）、ＮＫ－５２８（化学式７で表される化合物）、ＮＫ－５５７（化学式８で表される化合物）、ＮＫ－１５１６（化学式９で表される化合物）は、ＮＫ－４と同等以上の細胞増殖促進作用と、神経突起伸展促進作用を示した。また、細胞増殖促進活性があると評価した化合物について、アミロイドβフラグメントによる細胞傷害に対する抑制作用（評価法Ｃ）を調べたところ、いずれも、アミロイドβフラグメントによる細胞傷害に対する抑制作用が認められた。以上の結果、及び、実験１及び２の結果から、表６に示す細胞増殖促進作用や神経突起伸展促進作用を有する化合物は、いずれも、神経細胞を活性化しているので、ヒトの抗神経変性疾患剤として利用できることを物語っている。なかでも、ＮＫ－１９（化学式４で表される化合物）、ＮＫ－５３（化学式５で表される化合物）、ＮＫ－１００（化学式６で表される化合物）、ＮＫ－５２８（化学式７で表される化合物）、ＮＫ－５５７（化学式８で表される化合物）、ＮＫ－１５１６（化学式９で表される化合物）は、細胞増殖促進作用や神経突起伸展促進作用が強いので、特にヒトの抗神経変性疾患剤として有用であることを物語っている。さらに、これらの化合物、とりわけ、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、ＮＫ－５２８、ＮＫ－５５７、ＮＫ－１５１６は、ヒトの神経変性疾患及びそれに伴う病態や神経機能障害の治療剤としても利用できることを物語っている。また、表６において、アミロイドβフラグメントによる細胞傷害を抑制する作用を有する化合物は、アポトーシス抑制剤として有用であることを物語っている。 As shown in the results of Table 6, it was found that the 239 compounds tested had a cell growth promoting action (Evaluation Method A) and a neurite outgrowth promoting action (Evaluation Method B). In particular, NK-19 (compound represented by chemical formula 4), NK-53 (compound represented by chemical formula 5), NK-100 (compound represented by chemical formula 6), NK-528 (expressed by chemical formula 7) Compound), NK-557 (compound represented by chemical formula 8), and NK-1516 (compound represented by chemical formula 9) have cell growth promoting effects and neurite outgrowth promoting actions equivalent to or higher than those of NK-4. Indicated. Moreover, when the inhibitory effect (evaluation method C) with respect to the cytotoxicity by an amyloid (beta) fragment was investigated about the compound evaluated that it had cell growth promotion activity, all, the inhibitory effect with respect to the cytotoxicity by an amyloid (beta) fragment was recognized. Based on the above results and the results of Experiments 1 and 2, all of the compounds having the cell growth promoting action and the neurite outgrowth promoting action shown in Table 6 activate nerve cells. It shows that it can be used as a disease agent. Among them, NK-19 (compound represented by chemical formula 4), NK-53 (compound represented by chemical formula 5), NK-100 (compound represented by chemical formula 6), NK-528 (compound represented by chemical formula 7). Compound, NK-557 (compound represented by Chemical Formula 8), and NK-1516 (compound represented by Chemical Formula 9) have a strong cell growth promoting action and neurite outgrowth promoting action. It is useful as a neurodegenerative disease agent. Furthermore, these compounds, particularly NK-4, NK-19, NK-53, NK-100, NK-528, NK-557, NK-1516, are human neurodegenerative diseases and associated pathological and neurological dysfunctions. It can be used as a therapeutic agent. In addition, Table 6 shows that a compound having an action of suppressing cell damage caused by amyloid β fragment is useful as an apoptosis inhibitor.
＜実験４：各化合物の濃度がアミロイドβフラグメントによる細胞傷害に及ぼす影響＞
　実験３において、ＮＫ－４と同様にアミロイドβフラグメントによる細胞傷害を抑制する作用を持つことが確認されたＮＫ－１９、ＮＫ－５３、ＮＫ－１００、ＮＫ－５２８、ＮＫ－５５７及びＮＫ－１５１６について、その濃度が、アミロイドβフラグメントによる細胞傷害に及ぼす影響を調べた。すなわち、前記６種の化合物及びＮＫ－４を試験標品として使用し、各化合物が表７に示す終濃度となるように、ＰＣ１２－ＨＳ細胞を播種したウエルに添加した以外は、実験３の評価法Ｃと同一の条件で、これらの化合物のアミロイドβフラグメントによる細胞傷害に対する抑制効果を評価した。結果を細胞生存率（％）として表７に示す。 <Experiment 4: Effect of concentration of each compound on cell injury by amyloid β fragment>
In Experiment 3, NK-19, NK-53, NK-100, NK-528, NK-557, and NK-1516 were confirmed to have the effect of inhibiting cell damage caused by amyloid β fragment in the same manner as NK-4. , The effect of the concentration on cell damage by amyloid β fragment was examined. That is, except that the above 6 kinds of compounds and NK-4 were used as test preparations and each compound was added to the wells seeded with PC12-HS cells so as to have final concentrations shown in Table 7, the experiment 3 Under the same conditions as in Evaluation Method C, the inhibitory effect of these compounds on cytotoxicity by amyloid β fragment was evaluated. The results are shown in Table 7 as cell viability (%).
Figure JPOXMLDOC01-appb-T000053
Figure JPOXMLDOC01-appb-T000053
　表７から明らかなように、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、ＮＫ－５５７はＮＫ－４よりも低濃度で、アミロイドβフラグメントによる細胞傷害に対して、高い抑制活性を示した。このうち、最も低濃度で高い活性を示したのは、ＮＫ－５３で、１２．５ｎｇ／ｍｌの濃度でほぼ完全にアミロイドβフラグメントによる細胞傷害を抑制した（細胞生存率１１５±１６％）。ＮＫ－１９、ＮＫ－１００、ＮＫ－５５７では、５０ｎｇ／ｍｌの濃度で傷害抑制率が最高となり、それぞれ１０２±２７％、８８±１２％、１１４±９％となった。この効果は全て、ＮＫ－４を添加した場合に得られた細胞傷害抑制率（６９±７％）よりも高かった。ＮＫ－５３は、濃度が５０ｎｇ／ｍｌでも傷害抑制率が１１１±９％と高い抑制活性が認められた。一方、ＮＫ－４は２００ｎｇ／ｍｌの濃度で、傷害抑制率が１２２±３２％と最高になったのに対して、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、ＮＫ－５５７では細胞保護効果の低下が認められ、これらの化合物の細胞傷害抑制作用には至適濃度が存在することが確認され、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、ＮＫ－５５７の至適濃度は、ＮＫ－４より低いことが判明した。ＮＫ－５２８及びＮＫ－１５１６では、ＮＫ－４より高い細胞傷害抑制効果は認められなかった。以上の結果から、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、ＮＫ－５５７は、アルツハイマー病などの神経変性疾患に対してＮＫ－４より低濃度で、優れた効果を発揮する可能性が示された。また、ＮＫ－４よりも低濃度で、高い細胞傷害抑制活性の認められたＮＫ－１９及びＮＫ－１００は、他のものより分子量が大きく、ＮＫ１９やＮＫ－５３ではチアゾール環の窒素に結合した側鎖のアルキル基の炭素数が７（他は炭素数２）と多いことから、脂溶性が高く、細胞膜透過性が高いため、強い活性が発現されたと判断した。 As is clear from Table 7, NK-19, NK-53, NK-100, and NK-557 were at a lower concentration than NK-4, and showed high inhibitory activity against cell damage caused by amyloid β fragment. Among these, NK-53 showed the highest activity at the lowest concentration, and the cell damage by amyloid β fragment was almost completely suppressed at a concentration of 12.5 ng / ml (cell survival rate 115 ± 16%). In NK-19, NK-100, and NK-557, the injury inhibition rate was highest at a concentration of 50 ng / ml, which was 102 ± 27%, 88 ± 12%, and 114 ± 9%, respectively. All of these effects were higher than the cytotoxicity inhibition rate (69 ± 7%) obtained when NK-4 was added. NK-53 was found to have a high inhibitory activity of 111 ± 9% even at a concentration of 50 ng / ml. On the other hand, NK-4 had the highest injury inhibition rate of 122 ± 32% at a concentration of 200 ng / ml, whereas NK-19, NK-53, NK-100, and NK-557 had a cytoprotective effect. It was confirmed that there was an optimal concentration for the cytotoxic effect of these compounds, and the optimal concentrations of NK-19, NK-53, NK-100, and NK-557 are NK- It was found to be lower than 4. NK-528 and NK-1516 did not show a higher cytotoxic effect than NK-4. From the above results, it is shown that NK-19, NK-53, NK-100, and NK-557 may exhibit superior effects at lower concentrations than NK-4 for neurodegenerative diseases such as Alzheimer's disease. It was done. In addition, NK-19 and NK-100, which showed a high cytotoxicity-inhibiting activity at a lower concentration than NK-4, had a higher molecular weight than the others, and NK19 and NK-53 bound to nitrogen in the thiazole ring. Since the number of carbon atoms in the side chain alkyl group is as large as 7 (other carbon number is 2), it was judged that strong activity was expressed because of high lipid solubility and high cell membrane permeability.
＜実験５：アミロイドβペプチドの凝集に及ぼす影響＞
　実験３及び４において、アミロイドβフラグメントの細胞傷害性に対する保護作用が認められた化合物のなかから、神経突起伸展促進作用も確認されたＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７を試験試料に使用して、ヒトアルツハイマー病の治療剤開発の好適なモデルとされるアミロイドβペプチドの凝集に及ぼす影響を調べる試験を以下のように実施した。すなわち、各試験試料をＤＭＳＯ（ＳＩＧＭＡ社販売、カタログ番号「Ｄ８４１８」）に５ｍｇ／ｍｌの濃度で溶解した後、Ｍｉｌｌｅｘ－ＬＧ（Ｍｉｌｌｉｐｏｒｅ社販売、製品番号「ＬＬＧ０２５ＳＳ」、ＤＭＳＯ耐性）で膜ろ過し、Ｔｒｉｓ－ＨＣｌ緩衝液を使用して、２００ｎＭの濃度に調製して試験試料溶液とした。 <Experiment 5: Effect on Aggregation of Amyloid β Peptide>
In the experiments 3 and 4, NK-4, NK-19, NK-53, NK-100 and NK-4, NK-19, which were confirmed to have a neurite outgrowth promoting action among the compounds in which the protective action against the cytotoxicity of amyloid β fragment was recognized. Using NK-557 as a test sample, a test for examining the effect on aggregation of amyloid β peptide, which is a suitable model for developing a therapeutic agent for human Alzheimer's disease, was performed as follows. Specifically, each test sample was dissolved in DMSO (SIGMA, catalog number “D8418”) at a concentration of 5 mg / ml, and then membrane-filtered with Millex-LG (Millipore, product number “LLG025SS”, DMSO resistant). Using Tris-HCl buffer, the test sample solution was prepared to a concentration of 200 nM.
＜アミロイドβペプチド凝集の測定法＞
　アミロイドβペプチドの凝集はチオフラビンＴを用いた方法で測定した（例えば、Ｈｉｌａｌ　Ａ．Ｌａｓｈｕｅｌら、『Ｊｏｕｒｎａｌ　ｏｆ　Ｂｉｏｌｏｇｉｃａｌ　Ｃｈｅｍｉｓｔｒｙ』、第２７７巻、第４５号、第４２８８１－４２８９０頁（２００２年）参照）。チオフラビン－Ｔは、凝集したアミロイドβペプチドのβ－シート構造に結合し、蛍光を発する。この蛍光量を蛍光プレートリーダーで検出しアミロイドβペプチド凝集の指標とした。試験試料がアミロイドβペプチド凝集を抑制した場合、チオフラビン－Ｔの蛍光が減少する。この方法で試験試料のアミロイドβペプチド凝集に及ぼす影響を調べた。すなわち、配列表における配列番号２で表される４０個のアミノ酸からなるアミノ酸配列を有するヒトアミロイドβペプチドＡｎａ　Ｓｐｅｃ社販売）を、４００μＭの濃度に滅菌蒸留水に溶解して使用した。試験試料溶液は、Ｔｒｉｓ－ＨＣｌ緩衝液にて希釈した。反応用の容器に１００ｍＭ　アミロイドβペプチド１５μｌ及び試験試料溶液（２００ｎＭ）４５μｌを入れて、混合し、３７℃で６日間反応させた。反応後の溶液を５０μｌとり、１０μＭチオフラビンＴ４５０μｌと混合し、３０分後に、蛍光度計で測定した（励起波長４５０ｎｍ、吸収波長４８２ｎｍ）。チオフラビンＴのみの蛍光度を０％、１００ｍＭ　アミロイドβペプチド１５μｌ及びＴｒｉｓ－ＨＣｌ緩衝液４５μｌを入れ、混合し、３７℃で６日間反応させたときの蛍光度を１００％とし、各試験試料溶液を加えて反応させたときの蛍光強度の相対値を求め１００％から減じてアミロイドβペプチド凝集抑制効果（％）として表８に示す。 <Measurement method of amyloid β peptide aggregation>
Aggregation of amyloid β peptide was measured by a method using thioflavin T (see, for example, Hilal A. Laschel et al., “Journal of Biological Chemistry”, Vol. 277, No. 45, pp. 42881-42890 (2002)). . Thioflavin-T binds to the β-sheet structure of aggregated amyloid β peptide and emits fluorescence. This amount of fluorescence was detected with a fluorescence plate reader and used as an indicator of amyloid β peptide aggregation. When the test sample inhibits amyloid β peptide aggregation, the thioflavin-T fluorescence decreases. The effect of the test sample on amyloid β peptide aggregation was examined by this method. That is, a human amyloid β peptide having an amino acid sequence consisting of 40 amino acids represented by SEQ ID NO: 2 in the Sequence Listing (sold by Ana Spec) was used after dissolving in sterile distilled water at a concentration of 400 μM. The test sample solution was diluted with Tris-HCl buffer. In a reaction container, 15 μl of 100 mM amyloid β peptide and 45 μl of a test sample solution (200 nM) were added, mixed, and reacted at 37 ° C. for 6 days. 50 μl of the solution after the reaction was taken and mixed with 450 μl of 10 μM thioflavin T, and 30 minutes later, measurement was performed with a fluorometer (excitation wavelength 450 nm, absorption wavelength 482 nm). Fluorescence of only thioflavin T was 0%, 100 mM amyloid β peptide 15 μl and Tris-HCl buffer 45 μl were mixed, mixed, and reacted at 37 ° C. for 6 days. The fluorescence was 100%. In addition, the relative value of the fluorescence intensity when the reaction was carried out was determined and subtracted from 100% and shown in Table 8 as the amyloid β peptide aggregation inhibitory effect (%).
Figure JPOXMLDOC01-appb-T000054
Figure JPOXMLDOC01-appb-T000054
　表８から明らかなように、試験に使用したＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７はいずれも、アミロイドβペプチドの凝集を抑制し、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７はいずれも、ＮＫ－４よりも強い抑制活性を示した。なかでも、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７は、アミロイドβペプチドの凝集を９５％以上抑制し、ＮＫ－１００では９９％とほぼ完全に凝集を抑制した。この結果は、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７などの、アミロイドβフラグメントによる細胞傷害を抑制する作用を有する化合物は、アミロイドβペプチドの凝集を抑制する作用も有することから、アルツハイマー病の治療剤としてだけでなく、予防剤としても有用であることを物語っている。 As is clear from Table 8, all of NK-4, NK-19, NK-53, NK-100 and NK-557 used in the test suppressed aggregation of amyloid β peptide, and NK-19, NK- 53, NK-100 and NK-557 all showed stronger inhibitory activity than NK-4. Among them, NK-53, NK-100 and NK-557 suppressed amyloid β peptide aggregation by 95% or more, and NK-100 almost completely suppressed aggregation by 99%. As a result, a compound having an action of suppressing cell damage caused by amyloid β fragment such as NK-4, NK-19, NK-53, NK-100 and NK-557 has an action of suppressing aggregation of amyloid β peptide. Therefore, it shows that it is useful not only as a therapeutic agent for Alzheimer's disease but also as a preventive agent.
＜実験６：色素化合物の濃度の細胞増殖促進作用及び神経突起伸展作用に及ぼす影響＞
　実験４で、アミロイドβフラグメントによる細胞傷害に対する抑制効果があったものの中で、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、及び、ＮＫ－５５７を使用して、これら化合物の濃度が、ＰＣ１２－ＨＳ細胞の細胞増殖促進作用及び神経突起伸展作用に及ぼす影響を、実験３の評価法Ａ及びＢと同じ方法を用いて調べた。すなわち、細胞増殖促進は、実験３の評価法Ａと同様に、細胞を培養して、培地中の化合物の終濃度が、表９に示す濃度となるように、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、ＮＫ－５５７のいずれかを加えて培養を継続し、細胞をＡｌａｍａｒ　ｂｌｕｅで染色後、蛍光プレートリーダー（日本モレキュラーディバイス社販売）で５４４－５９０ｎｍの蛍光強度を測定した。化合物を含まない１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地のみを加えて培養したウエルの蛍光強度を１００％とした時の相対値を求めた。結果を細胞生存率（％）として表９に併せて示す。また、神経突起伸展は、実験３の評価法Ｂと同じ方法で細胞を培養し、培地中の化合物の終濃度が、表１０に示す濃度となるように、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、ＮＫ－５５７のいずれかを加えて培養を継続し、細胞をグルタルアルデヒドで固定した。一視野に約１００個の細胞を含む倍率で顕微鏡観察し、全細胞数に対する神経突起伸展の認められた細胞の割合（％）を求めた結果を表１０に示す。 <Experiment 6: Effect of pigment compound concentration on cell growth promoting action and neurite extension action>
Among those having an inhibitory effect on the cytotoxicity caused by amyloid β fragment in Experiment 4, using NK-19, NK-53, NK-100, and NK-557, the concentration of these compounds was changed to PC12- The effects of HS cells on the cell growth promoting action and neurite extension action were examined using the same methods as in Evaluation Methods A and B in Experiment 3. That is, cell growth is promoted by culturing cells in the same manner as in Evaluation Method A of Experiment 3, so that the final concentration of the compound in the medium becomes the concentration shown in Table 9, NK-4, NK-19, Culturing was continued by adding any of NK-53, NK-100, and NK-557. After staining the cells with Alamar blue, the fluorescence intensity at 544 to 590 nm was measured with a fluorescence plate reader (manufactured by Molecular Devices, Japan). . Relative values were determined when the fluorescence intensity of wells cultured with only 10% by volume FBS-added D-MEM medium containing no compound was taken as 100%. The results are also shown in Table 9 as cell viability (%). For neurite outgrowth, cells were cultured in the same manner as in Evaluation Method B of Experiment 3, and NK-4, NK-19, NK so that the final concentration of the compound in the medium was the concentration shown in Table 10. Either −53, NK-100, or NK-557 was added to continue the culture, and the cells were fixed with glutaraldehyde. Table 10 shows the results of determining the ratio (%) of cells in which neurite outgrowth was observed with respect to the total number of cells by microscopic observation at a magnification containing about 100 cells in one field of view.
Figure JPOXMLDOC01-appb-T000055
Figure JPOXMLDOC01-appb-T000055
Figure JPOXMLDOC01-appb-T000056
Figure JPOXMLDOC01-appb-T000056
　表９から明らかなように、培地に添加した化合物のうちＮＫ－４及びＮＫ－１００で特に強い細胞増殖促進作用が認められた。またこれらの化合物による細胞増殖促進作用には、至適濃度が認められ、ＮＫ－１９、ＮＫ－５３及びＮＫ－１００が、５０ｎｇ／ｍｌだったのに対して、ＮＫ－４及びＮＫ－５５７は、２００ｎｇ／ｍｌとなった。また、表１０から明らかなように、神経突起の伸展促進作用は、各化合物の濃度依存的に増加し、ＮＫ－１００で最も強い神経突起伸展促進作用が認められた。 As is clear from Table 9, NK-4 and NK-100 among the compounds added to the medium showed a particularly strong cell growth promoting action. In addition, the optimal concentration was observed for the cell growth promoting action by these compounds. NK-19, NK-53 and NK-100 were 50 ng / ml, whereas NK-4 and NK-557 were 200 ng / ml. Further, as apparent from Table 10, the neurite extension promoting action increased depending on the concentration of each compound, and the strongest neurite extension promoting action was observed with NK-100.
＜実験７：色素化合物の脳虚血モデルラットに及ぼす影響＞
　実験１乃至６の結果から、ＮＫ－４や、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、及び、ＮＫ－５５７には神経変性疾患に対する治療効果があると判断したので、これらの化合物の投与が、ヒトの脳梗塞の好適なモデルとして利用されている脳虚血モデルラットに及ぼす影響を、行動学的指標及び脳梗塞部位の体積を指標として調べた。 <Experiment 7: Effect of dye compound on cerebral ischemia model rat>
From the results of Experiments 1 to 6, it was determined that NK-4, NK-19, NK-53, NK-100, and NK-557 have a therapeutic effect on neurodegenerative diseases. However, the influence on the rat model of cerebral ischemia used as a suitable model of human cerebral infarction was examined using the behavioral index and the volume of the cerebral infarction site as an index.
＜脳虚血ラット＞
　ＳＤラット（雄性、７乃至８週齢、体重２８０乃至３３０ｇ、日本チャールスリバー株式会社販売）を、無作為に７群各５乃至７匹を割り付けた。これら７群のうち、５群を試験群として以下の手術を施した。予め、アトロピン（扶桑薬品、０．３ｍｇ／ｋｇ・体重）を皮下に投与した。次に、ウレタン（シグマ社販売６００ｍｇ／ｋｇ・体重）及びα－クロラロース（シグマ社販売、６０ｍｇ／ｋｇ・体重）を腹腔内に投与して、麻酔を施し、自然呼吸のまま、固定器に固定した。頚部正中切開を加え、迷走神経の保存に留意しつつ、右頚動脈分岐部に達した。右頚動脈分岐部を中心に、総頚動脈及び外頚動脈を周囲結合組織より剥離し、それぞれ６－０ナイロン糸（アルフレッサーファーマ株式会社販売、商品名「ネスコスーチャー」）にて結紮した。次に、内頚動脈に６－０ナイロン糸をかけ、塞栓挿入後の固定に備えた。次いで、総頚動脈を切開し、同部より先端をシリコンコートした４－０ナイロン糸で作成した塞栓（Ｄｏｃｃｏｌ）を内頚動脈に向けて約１６ｍｍ挿入し、総頚動脈にクリップで固定した。（例えば、小泉ら、「脳卒中」、第８巻、第１号、第１乃至８頁（１９８６年）参照）。なお、この方法により、塞栓のシリコンコートした先端部分は、中大脳動脈分岐部を越えて、前大脳動脈内に２ｍｍ程度入り、中大脳動脈入口を閉塞する。この状態のまま３７℃の保温パッド上で２時間中大脳動脈を閉塞した後、挿入した塞栓を抜き去り、血流を再開通（再灌流）させ、その後、総頚動脈切開部からの出血を防ぐため、内頚動脈を頚動脈分岐部近傍で結紮した。このモデルでの血流再開は、右総頚動脈が結紮されているため、左内径動脈及び、椎骨動脈、脳底動脈より、前・後交通動脈を介して行われる。 <Brain ischemic rat>
SD rats (male, 7 to 8 weeks old, body weight 280 to 330 g, sold by Nippon Charles River Co., Ltd.) were randomly assigned to 5 to 7 animals in 7 groups. Of these seven groups, the following surgery was performed with 5 groups as test groups. In advance, atropine (fuso medicine, 0.3 mg / kg · body weight) was subcutaneously administered. Next, urethane (sold by Sigma, 600 mg / kg, body weight) and α-chloralose (sold by Sigma, 60 mg / kg, body weight) were administered intraperitoneally, and anesthesia was applied. did. A midline cervical incision was made and the right carotid bifurcation was reached, paying attention to the preservation of the vagus nerve. The common carotid artery and the external carotid artery were peeled from the surrounding connective tissue, centering on the right carotid bifurcation, and ligated with 6-0 nylon thread (sold by Alfresa Pharma Co., Ltd., trade name “Nesscoacher”). Next, a 6-0 nylon thread was applied to the internal carotid artery to prepare for fixation after embolization. Next, the common carotid artery was incised, and an embolus (Doccol) made of 4-0 nylon thread coated with silicon at the tip was inserted into the internal carotid artery about 16 mm and fixed to the common carotid artery with a clip. (See, for example, Koizumi et al., “Stroke”, Vol. 8, No. 1, pp. 1-8 (1986)). By this method, the tip portion of the embolus that is silicon-coated enters the anterior cerebral artery beyond the middle cerebral artery bifurcation and closes the middle cerebral artery entrance. In this state, the cerebral artery is occluded for 2 hours on a thermal pad at 37 ° C., then the inserted embolus is removed, blood flow is resumed (reperfusion), and then bleeding from the common carotid artery incision is prevented. Therefore, the internal carotid artery was ligated near the carotid bifurcation. Since the right common carotid artery is ligated in this model, the blood flow is resumed from the left inner diameter artery, the vertebral artery, and the basilar artery via the anterior / posterior traffic artery.
＜化合物の投与＞
　試験に使用したＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７は、各々ＤＭＳＯ（ＳＩＧＭＡ社販売、商品番号「Ｄ８４１８」）に５ｍｇ／ｍｌの濃度で溶解した後、膜濾過（Ｍｉｌｌｉｐｏｒｅ社販売、商品名「Ｍｉｌｌｅｘ－ＬＧ　ＳＬＬＧ０２５ＳＳ」、ＤＭＳＯ耐性膜使用）した。各化合物を、使用時に、各々ＰＢＳに２５ｎｇ／ｍｌとなるように溶解して、そのいずれかを、５群各５匹又は７匹のラットに、中大脳動脈閉塞１時間後及び血液の再灌流時に尾静脈内に投与した（４ｍｌ／ｋｇ・体重、化合物の投与量１００μｇ／ｋｇ・体重）（試験群１乃至５）。血流再開２４時間後に、以下に述べる手法に基づいて行動学的及び組織学的評価を行った。残りの２群の内の１群５匹には、対照群１として、試験群１乃至５と同様の手術を施した後、化合物を含まないＰＢＳを４ｍｌ／ｋｇ・体重、中大脳動脈閉塞１時間後及び血液の再灌流時に尾静脈内投与した。また、残りの１群６匹には、対照群２として、総頚、外頚、内頚動脈の結紮した後、塞栓を中大脳動脈付近に挿入せずに、血流を再開させた偽手術（Ｓｈａｍ手術）を行った。対照群２にも、化合物を含まないＰＢＳを４ｍｌ／ｋｇ・体重、総頚、外頚、内頚動脈を結紮１時間後及び血液の再灌流時に尾静脈内投与した。対照群１及び２についても、試験群１乃至５と同様の行動学的及び組織学的評価を行った。 <Administration of compound>
NK-4, NK-19, NK-53, NK-100 and NK-557 used in the test were each dissolved in DMSO (SIGMA, product number “D8418”) at a concentration of 5 mg / ml, and then the membrane. Filtration (sold by Millipore, trade name “Millex-LG SLLG025SS”, using DMSO resistant membrane) was performed. Each compound was dissolved in PBS to 25 ng / ml at the time of use, and any one of them was administered to 5 or 7 rats in 5 groups each 1 hour after middle cerebral artery occlusion and blood reperfusion. Occasionally, it was administered into the tail vein (4 ml / kg · body weight, compound dose of 100 μg / kg · body weight) (test groups 1 to 5). 24 hours after resumption of blood flow, behavioral and histological evaluations were performed based on the method described below. Of the remaining 2 groups, 5 mice per group were treated with the same procedure as in test groups 1 to 5 as control group 1, followed by PBS containing no compound at 4 ml / kg · body weight, middle cerebral artery occlusion 1 It was administered via the tail vein after time and at the time of blood reperfusion. In the remaining 6 animals in 1 group, as control group 2, after ligation of the common neck, external neck, and internal carotid artery, sham operation was resumed without inserting an embolus near the middle cerebral artery ( (Sham operation) was performed. In the control group 2, PBS containing no compound was also administered into the tail vein at 4 ml / kg / body weight, the common neck, the external neck, and the internal carotid artery 1 hour after ligation and at the time of blood reperfusion. For control groups 1 and 2, behavioral and histological evaluations similar to those of test groups 1 to 5 were performed.
＜効果の評価方法＞
　＜行動学的及び組織学的評価＞
　＜行動学的評価＞
　表１１に示す基準に基づいて、各評価項目の症状の程度をスコア化し、個体ごとに積算して、行動学的スコアを求めた（Ｐｅｔｕｌｌｏ　Ｄ．等、『Ｌｉｆｅ　Ｓｃｉｅｎｃｅｓ』、第６４巻、第１３号、第１０９９乃至１１０８頁（１９９９年）参照）（最大スコア６）。結果を表１２に示す。
　＜組織学的評価＞
　試験終了後、各々のラットをエーテル麻酔下で、左心室からの生理食塩水を灌流させながら後大静脈を切断して脱血した。死後３分以内に脳を摘出し、スライサー（株式会社林原生物化学研究所製造）を使用して、冠状方向に、２ｍｍの厚さにスライスにした後、脳梗塞部位を、特異的に染色する２，３，５－ｔｒｉｐｈｅｎｙｌｔｅｔｒａｚｏｌｉｕｍ　ｃｈｌｏｒｉｄｅ（ＴＴＣ）を２質量／容積％含有するＰＢＳ中で３７℃、３０分間インキュベートし、１０容積％ホルマリン溶液中で１時間固定した（Ｂｅｎｄｅｒｓｏｎ　Ｊ．Ｂ．等，　『Ｓｔｒｏｋｅ』、第１７巻、第１３０４乃至１３０８頁（１９８６年）参照）。ＴＴＣで染色された脳梗塞部位の面積を、画像解析フリーソフト（Ｓｃｉｏｎ社販売、商品名「Ｓｃｉｏｎ　Ｉｍａｇｅ」）を用いて解析し、梗塞部位と脳全体の体積を算出した。梗塞部位の体積を、脳の全体積で除し、１００倍して、脳全体に占める梗塞部位の割合（％）を計算した。さらに、対照群１のラットの脳梗塞部位の脳全体に占める割合を１００％としたときの、試験群１乃至５のラットの脳梗塞部位の脳全体に占める割合の相対値を計算して、脳梗塞の大きさ（％）とした。結果を表１２に併せて示す。 <Evaluation method of effect>
<Behavioral and histological evaluation>
<Behavioral evaluation>
Based on the criteria shown in Table 11, the degree of symptom of each evaluation item was scored and accumulated for each individual to obtain a behavioral score (Petullo D. et al., “Life Sciences”, Vol. 64, No. 1). 13, pp. 1099 to 1108 (1999)) (maximum score 6). The results are shown in Table 12.
<Histological evaluation>
After completion of the test, each rat was exsanguinated by severing the posterior vena cava while perfusing physiological saline from the left ventricle under ether anesthesia. The brain is removed within 3 minutes after death, sliced to a thickness of 2 mm in the coronal direction using a slicer (produced by Hayashibara Biochemical Laboratories Co., Ltd.), and then the cerebral infarction site is specifically stained. 2,3,5-triphenyltetrazole chloride (TTC) was incubated in PBS containing 2% by mass / volume at 37 ° C. for 30 minutes and fixed in 10% by volume formalin solution for 1 hour (Benderson JB et al., “ Stroke, Vol. 17, pages 1304 to 1308 (1986)). The area of the cerebral infarction site stained with TTC was analyzed using image analysis free software (trade name “Scion Image”, sold by Scion), and the volume of the infarct site and the entire brain was calculated. The volume of the infarcted area was divided by the total volume of the brain, multiplied by 100, and the ratio (%) of the infarcted area to the entire brain was calculated. Furthermore, the relative value of the ratio of the cerebral infarction site of the rats of the test groups 1 to 5 to the entire brain when the ratio of the cerebral infarction site of the rats of the control group 1 to 100% is calculated, The size of cerebral infarction (%). The results are also shown in Table 12.
Figure JPOXMLDOC01-appb-T000057
Figure JPOXMLDOC01-appb-T000057
Figure JPOXMLDOC01-appb-T000058
Figure JPOXMLDOC01-appb-T000058
　表１２から明らかなように、偽手術を行ったラット（対照群２）の脳には梗塞部位は認められず、行動も全て正常であった。脳虚血を起こしたラット（対照群１）は、行動学的スコアが、５．８±０．１となって、評価した全ての項においてその運動能の殆どを喪失した。これに対して、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７のいずれかを投与したラット（試験群１乃至５）では、何れの場合にも、対照群１に比較して、運動能の喪失が有意に抑制された。脳梗塞の起こった部位の体積で見ても、脳虚血を起こしたラット（対照群１）と比較して、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７のいずれかを投与したラット（試験群１乃至５）では、何れの場合にも、脳梗塞を起こした部位が小さく、ＮＫ－４、ＮＫ－１９、ＮＫ－５３投与（試験群１乃至３）では、有意な脳梗塞部位の縮小が認められた。効果の強さを比較すると、ＮＫ－１９を投与した場合に、行動学的スコア及び脳梗塞部位の抑制率共に、最も強い改善が認められた。この結果は、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７は、虚血及びその後の血流再開が原因で引き起こされる神経変性及びそれに伴う神経機能障害に対する治療効果を有することを物語っている。 As is clear from Table 12, the infarcted area was not observed in the brains of rats subjected to sham surgery (control group 2), and the behavior was all normal. Rats with cerebral ischemia (control group 1) had a behavioral score of 5.8 ± 0.1 and lost most of their motility in all terms evaluated. On the other hand, in rats (test groups 1 to 5) administered with any of NK-4, NK-19, NK-53, NK-100 and NK-557, in any case, the control group 1 In comparison, loss of motor ability was significantly suppressed. In terms of the volume of the site where cerebral infarction occurred, NK-4, NK-19, NK-53, NK-100 and NK-557 were compared with those of rats (control group 1) that had cerebral ischemia. In either case (test groups 1 to 5), the site of cerebral infarction was small in any case, and in NK-4, NK-19, and NK-53 (test groups 1 to 3) A significant reduction in the cerebral infarction site was observed. When the strength of the effect was compared, the strongest improvement was observed in both the behavioral score and the inhibition rate of the cerebral infarction site when NK-19 was administered. This result shows that NK-4, NK-19, NK-53, NK-100 and NK-557 have a therapeutic effect on neurodegeneration caused by ischemia and the subsequent resumption of blood flow and the associated neurological dysfunction. Telling you to have.
＜実験８：色素化合物のアセチルコリンエステラーゼ（ＡｃｈＥ）活性に及ぼす影響＞
　アルツハイマー型認知症にはドネぺジルをはじめとするＡｃｈＥ阻害薬が臨床応用されている。ＡｃｈＥ阻害薬は中枢コリン神経系を賦活化し、虚血性認知症においても認知機能を改善することが報告されている。そこで、実験７で脳虚血を起こしたラットの脳梗塞及びそれに伴う神経機能障害を改善する効果が確認されたＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７に、ＡｃｈＥ阻害作用があるかどうかを確認する試験を行った。すなわち、ＤＭＳＯに溶解したＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７を、各々リン酸緩衝液で希釈し、濃度が表１３に示す濃度の１０倍濃度の化合物を含む溶液を調製して、試験試料溶液とした。実験１と同様に培養したＰＣ１２－ＨＳ細胞を回収し、その５倍量（容積）の１０ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝液（１Ｍ　ＮａＣｌ、５０ｍＭ　ＭｇＣｌ２、１％Ｔｒｉｔｏｎ　Ｘ－１００、ｐＨ７．２）を加えて、常法により均一にホモジナイズした後、４℃にて３０分間遠心分離（１０，０００ｇ）し、その上清を回収してアセチルコリンエステラーゼ（ＡｃｈＥ）含有溶液とした。９６ウエルプレート（住友ベークライト社販売、商品名「μテストプレート、細胞培養用、平底」）に、５０ｍＭリン酸緩衝液（ｐＨ８．０）３０μｌ、試験試料溶液１０μｌ、ＡｃｈＥ含有溶液１０μｌを入れ、さらに、反応基質液として０．５ｍＭヨウ化アセチルチオコリン（和光純薬工業株式会社販売）と１ｍＭ　２－ニトロ安息香酸（和光純薬工業株式会社販売）を含むリン酸緩衝液５０μｌを加えた。３７℃のインキュベーターで３０分間酵素反応を行った後、プレートリーダーで４０５ｎｍにおける吸光度（ＡＲ）を測定した。また、ＡｃｈＥ含有溶液の代わりにリン酸緩衝液１０μｌを用いて、上記と同様に３７℃のインキュベーターで３０分間酵素反応を行って吸光度（ＡＵ）を測定した。対照として、試験試料溶液に代えてリン酸緩衝液１０μｌを用いて同様に３７℃のインキュベーターで３０分間酵素反応を行い、吸光度（ＢＲ）を測定した。さらに、対照の対照として、対照におけるＡｃｈＥ含有液に代えてリン酸緩衝液１０μｌを用いて３７℃のインキュベーターで３０分間反応を行い、吸光度（ＢＵ）を測定した。以下の式によりＡｃｈＥ活性残存率（＝｛（ＡＲ－Ａｕ）÷（ＢＲ－ＢＵ）｝×１００）（％）を求め、ＡｃｈＥ活性を５０％阻害するときの化合物の濃度（ＩＣ５０）を求めた。結果を表１３に示す。 <Experiment 8: Effect of pigment compound on acetylcholinesterase (AchE) activity>
AchE inhibitors such as donepezil have been clinically applied to Alzheimer-type dementia. It has been reported that AchE inhibitors activate the central cholinergic nervous system and improve cognitive function even in ischemic dementia. Therefore, NK-4, NK-19, NK-53, NK-100, and NK-557, which were confirmed to have an effect of improving cerebral infarction and the accompanying neurological dysfunction in rats with cerebral ischemia in Experiment 7, A test was conducted to confirm whether there was an AchE inhibitory effect. That is, NK-4, NK-19, NK-53, NK-100 and NK-557 dissolved in DMSO were each diluted with a phosphate buffer, and a compound having a concentration 10 times that shown in Table 13 was added. A solution was prepared to provide a test sample solution. PC12-HS cells cultured in the same manner as in Experiment 1 were collected, and 5 volumes (volume) of 10 mM Tris-HCl buffer solution (1M NaCl, 50 mM MgCl 2 , 1% Triton X-100, pH 7.2) was added. After homogenizing uniformly by a conventional method, the mixture was centrifuged (10,000 g) at 4 ° C. for 30 minutes, and the supernatant was recovered to obtain a solution containing acetylcholinesterase (AchE). In a 96-well plate (commercially available from Sumitomo Bakelite Co., Ltd., trade name “μ test plate, for cell culture, flat bottom”), 30 μl of 50 mM phosphate buffer (pH 8.0), 10 μl of test sample solution, and 10 μl of AchE-containing solution were added. As a reaction substrate solution, 50 μl of a phosphate buffer containing 0.5 mM acetylthiocholine iodide (sold by Wako Pure Chemical Industries, Ltd.) and 1 mM 2-nitrobenzoic acid (sold by Wako Pure Chemical Industries, Ltd.) was added. After an enzyme reaction for 30 minutes in a 37 ° C. incubator, the absorbance (A R ) at 405 nm was measured with a plate reader. Further, using 10 μl of phosphate buffer instead of the AchE-containing solution, the enzyme reaction was carried out in a 37 ° C. incubator for 30 minutes in the same manner as described above, and the absorbance (A U ) was measured. As a control, an enzyme reaction was similarly carried out in a 37 ° C. incubator using 10 μl of phosphate buffer instead of the test sample solution, and the absorbance (B R ) was measured. Furthermore, as a control for the control, 10 μl of phosphate buffer was used instead of the AchE-containing solution in the control, and the reaction was performed in an incubator at 37 ° C. for 30 minutes, and the absorbance (B U ) was measured. The remaining ratio of AchE activity (= {(A R −A u ) ÷ (B R −B U )} × 100) (%) is determined by the following formula, and the concentration of the compound at which AchE activity is inhibited by 50% (IC 50 ). The results are shown in Table 13.
Figure JPOXMLDOC01-appb-T000059
Figure JPOXMLDOC01-appb-T000059
　表１３から明らかなように、低濃度域ではＮＫ－１００に最も高いＡｃｈＥ阻害が認められ、ＮＫ－１００の濃度が０．７８μｇ／ｍｌ以上で有意なＡｃｈＥ阻害作用を示した。また、ＮＫ－１００と構造の類似しているＮＫ－４でも３．１３μｇ／ｍｌ以上の濃度で有意な阻害が認められた。これらの化合物のＩＣ５０は、ＮＫ－４は３．３、ＮＫ－１００は、１１．８μｇ／ｍｌであった。これに対し、ＮＫ－１９及びＮＫ－５３のＡｃｈＥ阻害活性は弱く、ＮＫ－１９及びＮＫ－５３では２５μｇ／ｍｌの濃度でのみで有意な活性の低下が認められた。一方、ＮＫ－５５７では２５μｇ／ｍｌまでの濃度でほとんどＡｃｈＥ阻害活性を示さなかった。この結果は、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００の４種の化合物では、ＡｃｈＥの阻害作用によっても、コリン作動性神経系を賦活化し、アルツハイマー型認知症及び虚血性認知症を改善する可能性が示された。 As is clear from Table 13, NK-100 showed the highest AchE inhibition in the low concentration range, and showed a significant AchE inhibitory effect when the concentration of NK-100 was 0.78 μg / ml or more. In addition, NK-4 having a structure similar to that of NK-100 was significantly inhibited at a concentration of 3.13 μg / ml or more. The IC 50 of these compounds was 3.3 for NK-4 and 11.8 μg / ml for NK-100. In contrast, NK-19 and NK-53 had weak AchE inhibitory activity, and NK-19 and NK-53 showed a significant decrease in activity only at a concentration of 25 μg / ml. On the other hand, NK-557 showed almost no AchE inhibitory activity at concentrations up to 25 μg / ml. As a result, the four compounds NK-4, NK-19, NK-53, and NK-100 also activated the cholinergic nervous system by the inhibitory action of AchE, and caused Alzheimer-type dementia and ischemic cognition. The possibility of improving the symptoms was shown.
　なお、アルツハイマー病の治療薬として臨床利用されているガランタミンは、ＮＫ－４に比較して低い濃度で同程度のＡｃｈＥ阻害効果を示す（ガランタミンのＩＣ５０は、４４２μｇ／ｍｌ）。最近、これらの化合物では、ＮＫ－４と同様のＰＩ３Ｋ－Ａｋｔカスケードを介した細胞死の抑制も報告されているが、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７では数１００ｎｇ／ｍｌで効果が得られるのに対し、ドネぺジル、ガランタミン、タクリンでは同等の効果を得るために必要な化合物の濃度のオーダーが異なっている。また、Ｉｎ　ｖｉｔｒｏのアミロイドβフラグメント傷害モデルでの細胞保護効果についても同様なので、これらのことを考慮すると、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、ＮＫ－５５７は既存のアルツハイマー病治療薬とは異なる作用機作を有する抗神経変性疾患剤として利用できることを物語っている。 Note that galantamine, which is clinically used as a therapeutic agent for Alzheimer's disease, shows the same level of AchE inhibitory effect at a lower concentration than NK-4 (galantamine has an IC 50 of 442 μg / ml). Recently, these compounds have also been reported to suppress cell death via the PI3K-Akt cascade similar to NK-4, but NK-4, NK-19, NK-53, NK-100 and NK-557. The effect is obtained at several hundred ng / ml, whereas donepezil, galantamine, and tacrine differ in the order of the concentration of the compound necessary to obtain the same effect. In addition, since the cytoprotective effect in the in vitro amyloid β fragment injury model is the same, NK-4, NK-19, NK-53, NK-100, and NK-557 are considered to be existing Alzheimer's in view of these facts. It shows that it can be used as an anti-neurodegenerative disease agent having a mechanism of action different from that of a disease treatment drug.
＜実験９：色素化合物のフリーラジカルに及ぼす影響＞
　虚血後の血液の再灌流時の神経細胞傷害には、活性酸素種の関与が大きいといわれている。そこで、虚血性の神経細胞傷害の改善作用が認められた、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７に、ラジカル消去能があるかどうかを確認する試験を、フェントン反応によりジエチレントリアミン－Ｎ，Ｎ，Ｎ´，Ｎ´´，Ｎ´´－五酢酸（ＤＴＰＡ）から生じるヒドロキシラジカルに対する消去活性を電子スピン共鳴（以下、「ＥＳＲ」と略記する。）により測定することで行った。また、ぺルオキシラジカルは、不飽和脂肪酸とヒドロキシラジカルの反応によって生じる生体内脂質過酸化物の一種で、脂質含有率が高い脳は、ぺルオキシラジカルによる影響が大きいので、２，２´－アゾビス（２－アミジノプロパン）二塩酸塩（ＡＡＰＨ）の過熱により生じるぺルオキシラジカルの消去活性は、抗酸化成分が消去する生体由来ラジカルのモデルとされている。そこで、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７に、ペルオキシラジカル消去能があるかどうかを確認する試験を、ＡＡＰＨより生じるぺルオキシラジカルに対する消去活性をＥＳＲにより測定することで行った。 <Experiment 9: Effect of dye compound on free radical>
It is said that reactive oxygen species are largely involved in nerve cell damage during reperfusion of blood after ischemia. Therefore, a test for confirming whether NK-4, NK-19, NK-53, NK-100, and NK-557, which were found to have an effect of improving ischemic nerve cell injury, has a radical scavenging ability, The erasing activity for hydroxy radicals generated from diethylenetriamine-N, N, N ′, N ″, N ″ -pentaacetic acid (DTPA) by Fenton reaction is measured by electron spin resonance (hereinafter abbreviated as “ESR”). I went there. Peroxy radical is a kind of in vivo lipid peroxide generated by the reaction of unsaturated fatty acid and hydroxy radical. The brain with high lipid content is greatly affected by peroxy radical. —Peroxy radical scavenging activity caused by overheating of azobis (2-amidinopropane) dihydrochloride (AAPH) is a model of biologically derived radicals that are eliminated by antioxidant components. Therefore, a test for confirming whether NK-4, NK-19, NK-53, NK-100, and NK-557 have peroxy radical scavenging ability was conducted by using ESR to erase peroxy radicals generated from AAPH. This was done by measuring.
＜ヒドロキシラジカル消去能の測定方法＞
　ＤＭＳＯに溶解したＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７を、各々精製水で希釈し、濃度が表１４に示す濃度の３倍濃度の化合物を含む溶液を調製して、試験試料溶液とした。陽性対照として、フリーラジカル消去を作用機序とする脳保護剤エダラボン（田辺三菱製薬株式会社販売、商品名「ラジカット」、有効成分：３－メチル－１－フェニル－２－ピラゾリン－５－オン）を、表１４に示す濃度の３倍濃度に精製水で希釈して使用した。精製水５０μｌに８９ｍＭ　５，５－ジメチル－１－プロリン－オキシド（ＤＭＰＯ、同仁化学研究所販売）５０μｌ、試験試料溶液５０μｌ、１ｍＭ過酸化水素水、及び、１ｍＭ　ＦｅＳＯ４と１ｍＭ　ＤＴＰＡ（和光純薬工業株式会社販売）を含む水溶液５０μｌを加え、ボルテックスミキサーで１０秒間混合した後、３７℃の恒温槽中で４０秒間反応させた反応液を、反応終了３０秒後にＥＳＲ測定に使用した。試験試料溶液に代えてエダラボンの希釈液５０μｌを用いたこと以外同様にして反応をさせ、ＥＳＲ測定に供した。 <Measurement method of hydroxy radical scavenging ability>
NK-4, NK-19, NK-53, NK-100 and NK-557 dissolved in DMSO are each diluted with purified water to prepare a solution containing a compound having a concentration 3 times the concentration shown in Table 14. Thus, a test sample solution was obtained. As a positive control, edaravone, a brain protective agent with the action mechanism of free radical scavenging (sold by Mitsubishi Tanabe Pharma Corporation, trade name “Radicut”, active ingredient: 3-methyl-1-phenyl-2-pyrazolin-5-one) Was diluted with purified water to a concentration 3 times that shown in Table 14 and used. 50 μl of purified water, 50 μl of 89 mM 5,5-dimethyl-1-proline-oxide (DMPO, sold by Dojindo Laboratories), 50 μl of test sample solution, 1 mM hydrogen peroxide, and 1 mM FeSO 4 and 1 mM DTPA (Wako Pure Chemical Industries, Ltd.) After adding 50 μl of an aqueous solution containing Kogyo Kogyo Co., Ltd. and mixing for 10 seconds with a vortex mixer, the reaction solution reacted for 40 seconds in a constant temperature bath at 37 ° C. was used for ESR measurement 30 seconds after completion of the reaction. The reaction was conducted in the same manner except that 50 μl of edaravone diluted solution was used instead of the test sample solution, and subjected to ESR measurement.
＜ペルオキシラジカル消去能の測定方法＞
　ＤＭＳＯに溶解したＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７を、各々０．１Ｍリン酸緩衝液で希釈し、濃度が表１５に示す濃度の３倍濃度の化合物を含む溶液を調製して、試験試料溶液とした。０．１Ｍリン酸緩衝液（ｐＨ７．４）５０μｌに、１８０ｍＭ　５，５－ジメチル－１－プロリン－オキシド（ＤＭＰＯ、同仁化学研究所販売）５０μｌ、試験試料溶液５０μｌ、１００ｍＭ　ＡＡＰＨ（和光純薬工業株式会社販売）５０μｌを加え、ボルテックスミキサーで１０秒間混合した後、３７℃の恒温槽中で２分５０秒間反応させた反応液を、反応終了３０秒後にＥＳＲ測定に使用した。 <Measurement method of peroxy radical scavenging ability>
NK-4, NK-19, NK-53, NK-100 and NK-557 dissolved in DMSO are each diluted with 0.1 M phosphate buffer, and the concentration is a compound having a concentration three times that shown in Table 15. A solution containing was prepared as a test sample solution. 50 μl of 0.1 M phosphate buffer (pH 7.4), 50 μl of 180 mM 5,5-dimethyl-1-proline-oxide (DMPO, sold by Dojindo Laboratories), 50 μl of test sample solution, 100 mM AAPH (Wako Pure Chemical Industries, Ltd.) 50 μl was added and mixed with a vortex mixer for 10 seconds, and then the reaction solution reacted for 2 minutes and 50 seconds in a constant temperature bath at 37 ° C. was used for ESR measurement 30 seconds after the completion of the reaction.
＜ＥＳＲ測定法＞
　ヒドロキシラジカル測定用又はペルオキシラジカル測定用の反応液をＥＳＲ用扁平石英セルにとり、電子スピン共鳴（ＥＳＲ）装置（日本電子株式会社販売、商品名「Ｆｒｅｅ　ｒａｄｉｃａｌ　Ｍｏｎｉｔｏｒ　ＪＥＳ－ＦＲ３０」）にセットし、マニュアルに従ってＥＳＲを測定した。試験試料溶液に代えて、ヒドロキシラジカル測定の場合には精製水を、ペルオキシラジカル測定の場合には０．１Ｍリン酸緩衝液を混合して、試験試料溶液を加えて反応させたときと同様に反応させて、ＥＳＲを測定したときの値を１００として、試験試料溶液を加えて反応させたときの相対強度を求め、ヒドロキシラジカルの残存率（％）及びペルオキシラジカルの残存率（％）として、表１４及び１５にそれぞれ示す。さらに、これらの結果をもとに、各化合物のラジカル消去能のＩＣ５０を求めた結果を表１４及び１５に併せて示す。なお、ヒドロキシラジカル消去能の測定で陽性対照として使用したエダラボンは、２５μｇ／ｍｌ以下の濃度ではラジカル消去能が全く認められなかったので、さらに高濃度条件で試験を実施してＩＣ５０を求めた結果を表１４に併せて示した。また、この時のＥＳＲ測定条件は以下のように設定した。
　＜測定条件＞
　Ｐｏｗｅｒ　　　　　　　　　　　　：４ｍＷ
　Ｍａｇｎｅｔｉｃ　ｆｉｅｌｄ　　　：３３５．５ｍＴ
　Ｓｗｅｅｐ　ｔｉｍｅ　　　　　　　：２分
　Ｍｏｄｕｌａｔｉｏｎ　ｗｉｄｔｈ　：０．０７９ｍＴ
　Ａｍｐｌｉｔｕｄｅ　　　　　　　　：７９（ヒドロキシラジカルの測定の場合）、
　　　　　　　　　　　　　　　　　　　１２５（ペルオキシラジカルの測定の場合）
　Ｔｉｍｅ　ｃｏｎｓｔａｎｔ　　　　：０．１秒
　Ａｃｃｕｍ　　　　　　　　　　　　：１ <ESR measurement method>
The reaction solution for hydroxy radical measurement or peroxy radical measurement is placed in a flat quartz cell for ESR and set in an electron spin resonance (ESR) apparatus (trade name “Free radical Monitor JES-FR30” sold by JEOL Ltd.). ESR was measured according to Instead of the test sample solution, purified water is used in the case of hydroxy radical measurement, and 0.1 M phosphate buffer is mixed in the case of peroxy radical measurement, and the test sample solution is added and reacted in the same manner. React and set the value when measuring ESR as 100, determine the relative strength when the test sample solution was added and reacted, and as the residual rate of hydroxy radicals (%) and the residual rate of peroxy radicals (%), They are shown in Tables 14 and 15, respectively. Furthermore, based on these results, the results of determining the IC 50 of the radical scavenging ability of each compound are shown in Tables 14 and 15. In addition, since edaravone used as a positive control in the measurement of hydroxy radical scavenging ability showed no radical scavenging ability at a concentration of 25 μg / ml or less, the test was further performed under higher concentration conditions to obtain IC 50 . The results are also shown in Table 14. Moreover, the ESR measurement conditions at this time were set as follows.
<Measurement conditions>
Power: 4mW
Magnetic field: 335.5 mT
Sweep time: 2 minutes Modulation width: 0.079 mT
Amplitude: 79 (for measurement of hydroxy radical),
125 (when measuring peroxy radicals)
Time constant: 0.1 seconds Accum: 1
Figure JPOXMLDOC01-appb-T000060
Figure JPOXMLDOC01-appb-T000060
Figure JPOXMLDOC01-appb-T000061
Figure JPOXMLDOC01-appb-T000061
　表１４から明らかなように、試験試料液を混合して反応させたときの、ヒドロキシラジカル残存率は、試験試料液に含まれる化合物の濃度に依存して低下し、添加した化合物には、いずれも、ヒドロキシラジカルを消去する活性が確認された。また、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７のヒドロキシラジカル消去能のＩＣ５０は、いずれも６μｇ／ｍｌ前後だったのに対して、市販のフリーラジカル消去を作用機序とする脳保護剤エダラボンは１４８μｇ／ｍｌだったことから、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７は、市販の脳保護用のフリーラジカル消去剤よりも優れたヒドロキシラジカル消去能を有することが明らかになった。また、同様の方法で測定したＮＫ－９６９４及びＮＫ－１５０のヒドロキシラジカル消去能のＩＣ５０は、各々３．４μｇ／ｍｌ及び１．８μｇ／ｍｌであった。また、表１５から明らかなように、試験試料液を混合して反応させたときの、ペルオキシラジカル残存率は、試験試料液に含まれる化合物の濃度に依存して低下し、添加した化合物には、いずれも、ペルオキシラジカルを消去する活性が確認された。特に、ＮＫ－４を添加した場合には、高いラジカル消去活性が認められ、このラジカル消去能は、同濃度のアスコルビン酸ナトリウム（ＡｓＡ－Ｎａ）のもつ消去能とほぼ同等であった（データは示していない）。次に、ＮＫ－１９及びＮＫ－５３に高い活性が認められ、５μｇ／ｍｌ以上の濃度で有意なラジカル消去活性を示した。ＮＫ－５５７及びＮＫ－１００では、それぞれ２５μｇ／ｍｌ及び５０μｇ／ｍｌ以上の濃度では有意にペルオキシラジカルを消去した。以上の結果より、試験に使用した５種類の化合物はいずれも、強いヒドロキシラジカル消去能を有し、ＮＫ－４、ＮＫ－１９、ＮＫ－５３の３種類の化合物は、比較的低濃度でもペルオキシラジカル消去活性も示すことから、これらの化合物の脳梗塞のモデルラットにおける梗塞縮小効果の主要な作用メカニズムの一つにヒドロキシラジカルやペルオキシラジカルなどのフリーラジカル消去能が関与していると考えられた。現に、実験７では、ＮＫ－４、ＮＫ－１９、ＮＫ－５３投与群の方が、ＮＫ－５５７及びＮＫ－１００投与群よりも、高い梗塞縮小効果が認められていることから、これらの化合物は、虚血性疾患の血液の再灌流時のヒドロキシラジカル及び／又はペルオキシラジカルによる酸化ストレスを抑制し、神経細胞死を抑制する作用があり、抗神経変性疾患剤、脳保護剤、ラジカルスカベンジャー、酸化ストレス抑制剤、過酸化脂質生成抑制剤、脳の酸化的障害抑制剤などとして有効であることを物語っている。 As is clear from Table 14, the residual ratio of hydroxy radicals when the test sample solution is mixed and reacted decreases depending on the concentration of the compound contained in the test sample solution. Also, the activity of scavenging hydroxy radicals was confirmed. Further, the IC 50 of hydroxy radical scavenging ability of NK-4, NK-19, NK-53, NK-100 and NK-557 was all around 6 μg / ml, whereas commercially available free radical scavenging was not performed. Since the brain protective agent edaravone as the mechanism of action was 148 μg / ml, NK-4, NK-19, NK-53, NK-100 and NK-557 are more than commercially available free radical scavengers for brain protection. It was also revealed that it has excellent hydroxy radical scavenging ability. Further, IC 50 of hydroxy radical scavenging ability of NK-9694 and NK-150 measured by the same method were 3.4 μg / ml and 1.8 μg / ml, respectively. Further, as apparent from Table 15, the peroxy radical residual rate when the test sample solution was mixed and reacted was lowered depending on the concentration of the compound contained in the test sample solution, In both cases, the activity of scavenging peroxy radicals was confirmed. In particular, when NK-4 was added, a high radical scavenging activity was observed, and this scavenging ability was almost equivalent to that of sodium ascorbate (AsA-Na) at the same concentration (data is shown) Not shown). Next, NK-19 and NK-53 showed high activity, and showed significant radical scavenging activity at a concentration of 5 μg / ml or more. In NK-557 and NK-100, peroxy radicals were significantly eliminated at concentrations of 25 μg / ml and 50 μg / ml or more, respectively. From the above results, all five compounds used in the test have strong hydroxy radical scavenging ability, and the three compounds NK-4, NK-19, and NK-53 are peroxyl even at relatively low concentrations. Since it also exhibits radical scavenging activity, it was considered that free radical scavenging ability such as hydroxy radicals and peroxy radicals was involved in one of the main action mechanisms of infarct reduction effect in model rats with cerebral infarction. . In fact, in Experiment 7, the NK-4, NK-19, and NK-53 administration groups showed higher infarct reduction effects than the NK-557 and NK-100 administration groups. Suppresses oxidative stress caused by hydroxy radicals and / or peroxy radicals during reperfusion of blood in ischemic diseases, and suppresses neuronal cell death, anti-neurodegenerative disease agent, brain protective agent, radical scavenger, oxidation It demonstrates its effectiveness as a stress suppressor, lipid peroxide production inhibitor, brain oxidative disorder inhibitor, and the like.
　一方、陽性対照として使用したエダラボンは、フリーラジカル消去能（フリーラジカルスカベンジャー）を有し、ラット脳虚血モデルにおいて血液の再灌流後の、脳内のヒドロキシラジカルの生成を抑制すると共に、脳梗塞巣の進展、脳梗塞周辺領域血流量低下抑制作用、脳浮腫の抑制及び遅発性神経細胞死を抑制する作用などを有していることが知られている（例えば、『日本薬理学雑誌』、第１１９巻、第３０１乃至３０８頁（２００２年）参照）。これに対して、本発明の抗神経変性疾患剤は、エダラボンよりも強いヒドロキシラジカル消去能を有し、脳梗塞の進展抑制作用や神経細胞死抑制作用を有していることが判明したので、エダラボンと同等又はこれ以上に、脳保護剤、虚血性の脳障害の抑制剤、脳梗塞の進展抑制剤、脳浮腫の抑制剤及び遅発性神経死抑制剤などとして有利に利用できることを物語っている。また、エダラボンの有効成分である３－メチル－１－フェニル－２－ピラゾリン－５－オンやその類縁体は、そのラジカルスカベンジャーや過酸化脂質生成抑制作用を利用して、前記以外にも、脳機能正常化剤（特公平５－３１５２３号公報）、過酸化脂質生成抑制剤（特公平５－３５１２８号公報）、抗潰瘍剤（特許第２９０６５１２号公報）、血糖上昇抑制剤（特許第２９０６５１３号公報）、白内障や角膜障害などの眼性疾患の予防・治療剤（特開平７－２５７６５号公報、特開２００８－２６６１４２号公報）、移植臓器保存剤（特開平９－５２８０１号公報、国際公開ＷＯ０３／６７９７９号パンフレット）や移植組織（皮膚を含む）・臓器の壊死防止剤（特開平１１－７９９９１号公報）、急性腎不全・薬物などによる腎障害、皮膚組織障害、肺障害、肝繊維化・化学物質・エンドトキシン・虚血などによる肝障害、熱傷などによる皮膚組織の障害、脊髄損傷、脳内血管や動脈などの血管障害、心筋などの筋肉障害、尿細管間質障害などの各種臓器の障害やそれに伴う機能障害の治療・予防剤（特開平９－５２８３１号公報、特開２００４－９９５６０号公報、特開平１０－２７９４８０号公報、特開２００４－１３１４０２号公報、特開２００６－９６６６４号公報、特開２００４－１２３７１６号公報、特開２００４－２３８１号公報、特開２００４－１１５５０８号公報、国際公開ＷＯ０３／１０５９０９号パンフレット、国際公開ＷＯ０４／１３１０７号パンフレット、特開２００４－６７５８５号公報、特開２００４－１１５５０５号公報、特開２００８－５０９８７９号公報、国際公開ＷＯ０３／６６０５１号パンフレット、国際公開ＷＯ０３／８０５８３号パンフレット、特開２００６－１８２６７７号公報）、放射線障害予防剤・治療剤（特開２００３－３３５６７４号公報）、抗腫瘍剤・腫瘍転移抑制剤（特開２００４－２７７３１５号公報、２００５－２９５７３号公報）、細胞障害マーカー抑制剤（特開２００４－１３７２５２号公報）、心筋炎、膵臓炎、腸炎、関節、アレルギーをはじめとする各種組織や臓器の炎症性疾患やそれに伴う組織障害の予防・治療剤（特開２００４－１３７２５３号公報、特開２００４－１４３１４９号公報、国際公開ＷＯ０４／２２５４３号パンフレット、国際公開ＷＯ０５／１２２５５号公報）、視細胞障害、視神経障害、網膜疾患、聴覚細胞障害、聴覚神経障害などの感覚細胞、感覚神経或いは感覚器の障害抑制剤（国際公開ＷＯ０２／２６０号パンフレット、特開２００３－２５２７６０号公報、特開２００４－１２３７１３号公報、特開２００４－１３７２５６号公報）、パラコートなどの薬物中毒の予防・治療剤（特開２００４－１６１７２０号公報）、カルシウム・ナトリウム交換系阻害剤（特開２００４－１１５５１１号公報）、酸化ストレス抑制剤（国際公開ＷＯ０３／２４４４６号パンフレット）、疼痛や掻痒の予防・治療剤（特開２００４－３３１６５３号公報、特開２００８－３７７５３号公報）、プロテインキナーゼ刺激剤（特開２００４－３３９２１４号公報）、ミトコンドリア脳筋症予防・治療剤（特開２００５－８９４５６号公報）、動脈閉塞・狭窄予防・治療剤（特開２００５－１６２７４９号公報）、血液脳関門破綻抑制剤（国際公開ＷＯ０４／６３１６７号パンフレット）、薬物依存症治療剤（特開２００８－２４７８１３号公報）、アポトーシス抑制剤（特開２００３－３００８８０号公報）などとしても利用できることも知られているので、本発明の抗神経変性疾患剤は、エダラボンと同等又はこれ以上に優れた効果を有するこれらの疾患の予防・治療剤として有利に利用できることを物語っている。 On the other hand, edaravone used as a positive control has free radical scavenging ability (free radical scavenger), suppresses the generation of hydroxy radicals in the brain after blood reperfusion in a rat cerebral ischemia model, and cerebral infarction It is known to have nest development, blood flow reduction inhibitory effect around the cerebral infarction, brain edema and delayed neuronal cell death (for example, “The Japanese Pharmacology Journal” 119, 301-308 (2002)). On the other hand, the anti-neurodegenerative disease agent of the present invention has a stronger hydroxy radical scavenging ability than edaravone, and it has been found that it has a cerebral infarction progression inhibitory action and a nerve cell death inhibitory action. It tells us that it can be advantageously used as a brain protective agent, an ischemic brain disorder inhibitor, a cerebral infarction progression inhibitor, a brain edema inhibitor, and a delayed neuronal death inhibitor equivalent to or better than edaravone. Yes. In addition to the above, 3-methyl-1-phenyl-2-pyrazolin-5-one, which is an active ingredient of edaravone, and its analogs, use its radical scavenger and lipid peroxide production inhibitory action, in addition to the above. Function normalizing agent (Japanese Patent Publication No. 5-31523), lipid peroxide production inhibitor (Japanese Patent Publication No. 5-35128), anti-ulcer agent (Patent No. 2906512), blood sugar elevation inhibitor (Patent No. 2906513) ), Preventive / therapeutic agents for ophthalmic diseases such as cataracts and corneal disorders (JP-A-7-25765, JP-A-2008-266142), transplanted organ preservative (JP-A-9-52801, international publication) WO03 / 67979 pamphlet), transplanted tissue (including skin), organ necrosis inhibitor (Japanese Patent Laid-Open No. 11-79991), acute renal failure, renal damage due to drugs, Skin tissue disorders, lung disorders, liver disorders due to liver fibrosis, chemical substances, endotoxin, ischemia, etc., skin tissue disorders due to burns, spinal cord injury, vascular disorders such as intracerebral blood vessels and arteries, muscle disorders such as myocardium, Therapeutic / preventive agents for various organ disorders such as tubulointerstitial disorders and associated functional disorders (Japanese Patent Laid-Open Nos. 9-52831, 2004-99560, 10-279480, 2004) 131402, JP-A-2006-96664, JP-A-2004-123716, JP-A-2004-2381, JP-A-2004-115508, International Publication WO03 / 105909, International Publication WO04 / 13107 Pamphlet, JP 2004-67585 A, JP 2004-115505 A, JP 2008- No. 09879, International Publication No. WO03 / 66051, International Publication No. WO03 / 80583, Japanese Patent Laid-Open No. 2006-182676, Radiation Disorder Prevention / Treatment Agent (Japanese Patent Laid-Open No. 2003-335684), Antitumor Agent Tumor metastasis inhibitor (Japanese Patent Application Laid-Open Nos. 2004-277315 and 2005-29573), cytopathic marker inhibitor (Japanese Patent Application Laid-Open No. 2004-137252), myocarditis, pancreatitis, enteritis, joints, and allergies Preventive / therapeutic agents for inflammatory diseases of various tissues and organs and associated tissue disorders (JP 2004-137253 A, JP 2004-143149 A, International Publication WO 04/22543 Pamphlet, International Publication WO 05/12255 Publication) ), Photoreceptor cell disorder, optic neuropathy, retinal disease, auditory cell disorder, Sensory cell disorders such as auditory neuropathy, sensory nerve or sensory organ disorder inhibitors (International Publication WO02 / 260, JP2003-252760, JP2004-123713, JP2004-137256) , Preventive and therapeutic agents for drug addiction such as paraquat (JP 2004-161720 A), calcium / sodium exchange system inhibitors (JP 2004-115511 A), oxidative stress inhibitors (International Publication WO 03/24446 pamphlet) ), Preventive / therapeutic agents for pain and pruritus (Japanese Patent Application Laid-Open No. 2004-331653, Japanese Patent Application Laid-Open No. 2008-37753), protein kinase stimulator (Japanese Patent Application Laid-Open No. 2004-339214), preventive / therapeutic agent for mitochondrial encephalomyopathy (Japanese Patent Laid-Open No. 2005-89456), arterial occlusion / stenosis prevention / treatment Agent (Japanese Patent Laid-Open No. 2005-162749), blood brain barrier breakdown inhibitor (International Publication WO04 / 63167 pamphlet), drug dependence treatment agent (Japanese Patent Laid-Open No. 2008-247813), apoptosis inhibitor (Japanese Patent Laid-Open No. 2003-24713) It is also known that the anti-neurodegenerative disease agent of the present invention can be advantageously used as a prophylactic / therapeutic agent for these diseases having an effect equivalent to or better than that of edaravone. It tells you what you can do.
＜実験１０：ＮＫ－１９類縁体の神経栄養因子様活性＞
　上記実験で、強い神経変性抑制作用が確認されたＮＫ－１９について、その類縁体にも同様の作用があることを確認する試験をおこなった。すなわち、下記一般式３で表される化合物において、側鎖のアルキル基（Ｒ７乃至Ｒ９）の炭素数が１乃至１２で、対アニオンがＩ－又はＣｌ－である１２種類の化合物を合成（株式会社林原生物化学研究所合成）し、実験３と同様に、ＰＣ１２－ＨＳ細胞に対する細胞増殖促進作用及び神経突起伸展促進作用の強さを調べた。表１６に示すＮＫ－１９を含む、ＮＫ－１９の類縁体１２種類の化合物を、各々ＤＭＳＯに５ｍｇ／ｍｌとなるように溶解した。この溶液を１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈して、化合物の濃度が、各々１００ｎｇ／ｍｌ又は２μｇ／ｍｌとなる試験試料溶液を調製した。また、ＮＫ－２４及びＮＫ－１９については、その対アニオンをＩ－からＣｌ－に替えた化合物（ＮＫ－５６及びＮＫ－５３）も、ＤＭＳＯに５ｍｇ／ｍｌとなるように溶解した。これらの溶液を１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈して、化合物の濃度を、各々１００ｎｇ／ｍｌ又は２μｇ／ｍｌに希釈して試験試料溶液を調製した。 <Experiment 10: Neurotrophic factor-like activity of NK-19 analog>
In the above experiment, a test was conducted to confirm that NK-19, which has been confirmed to have a strong neurodegeneration inhibitory effect, has similar effects on its analogs. That is, in the compounds represented by the following general formula 3, the number of carbon atoms in the alkyl group of the side chain (R 7 to R 9) is 1 to 12, counter anion I - or Cl - the a is 12 kinds of compound synthesis (Synthesis by Hayashibara Biochemical Laboratories Co., Ltd.), and in the same manner as in Experiment 3, the strength of the cell growth promoting action and neurite extension promoting action on PC12-HS cells was examined. Twelve types of NK-19 analogs including NK-19 shown in Table 16 were dissolved in DMSO to a concentration of 5 mg / ml. This solution was diluted with 10% by volume FBS-added D-MEM medium to prepare a test sample solution having a compound concentration of 100 ng / ml or 2 μg / ml, respectively. Moreover, the NK-24 and NK-19 is the counter anion I - Cl from - compounds instead (NK-56 and NK-53) was also dissolved at a 5 mg / ml in DMSO. These solutions were diluted with 10% by volume FBS-added D-MEM medium to prepare test sample solutions by diluting the compound concentration to 100 ng / ml or 2 μg / ml, respectively.
＜細胞増殖促進作用の評価＞
　実験３の評価法Ａと同様に、コラーゲンコートした９６ウエルプレートに５×１０３個／ウエルになるように１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈し、１００μｌ／ウエルで播種した。２４時間後に１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈し、化合物の濃度を１００ｎｇ／ｍｌに調整した各試験試料溶液を１００μｌ／ウエル添加し、３７℃、５容積％ＣＯ２インキュベーター内で３日間培養した。培養３日目に培養上清を除去し、１０質量％Ａｌａｍａｒ　ｂｌｕｅ（Ｔｒｅｋ　Ｄｉａｇｎｏｓｔｉｃ）／１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地を２００μｌ／ウエルずつ添加し、６時間、３７℃、５容積％ＣＯ２インキュベーター中で培養し、蛍光プレートリーダー（日本モレキュラーディバイス株式会社販売）で５４４－５９０ｎｍの蛍光強度を測定した。各試験試料溶液を添加した場合の細胞増殖促進作用は、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地を１００μｌ／ウエル添加した対照の値を１００とした時の相対値として求めた。結果を表１６に示す。なお、試験は、各試験試料溶液につき、トリプレットで２回実施し、その平均を求めた。 <Evaluation of cell proliferation promoting action>
In the same manner as in Evaluation Method A in Experiment 3, a 96-well plate coated with collagen was diluted with 10% by volume FBS-added D-MEM medium to 5 × 10 3 cells / well and seeded at 100 μl / well. After 24 hours, each test sample solution diluted with 10% by volume FBS-added D-MEM medium and adjusted to a compound concentration of 100 ng / ml was added at 100 μl / well, and the mixture was kept at 37 ° C. in a 5% by volume CO 2 incubator for 3 days. Cultured. On the third day of culture, the culture supernatant was removed, and 10% by weight Alamar blue (Trek Diagnostics) / 10 volume% FBS-added D-MEM medium was added at 200 μl / well for 6 hours at 37 ° C., 5 volume% CO 2. After culturing in an incubator, the fluorescence intensity at 544 to 590 nm was measured with a fluorescence plate reader (available from Nippon Molecular Devices Co., Ltd.). The cell growth promoting effect when each test sample solution was added was determined as a relative value when the value of the control added with 100 μl / well of 10% by volume FBS-added D-MEM medium was taken as 100. The results are shown in Table 16. In addition, the test was implemented twice with each triplet for each test sample solution, and the average was obtained.
＜神経突起伸展促進作用の評価＞
　実験３の評価Ｂと同様に、ＰＣ１２－ＨＳ細胞を、予めコラーゲンコートした９６ウエルマイクロプレートに５×１０３個／ウエルになるように１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈し、１００μｌ／ウエルで播種した。２４時間後に、化合物の濃度を２μｇ／ｍｌに調整した各試験試料溶液５０μｌ／ウエルとＮＧＦ（終濃度５ｎｇ／ｍｌ）５０μｌ／ウエルとを添加し、培養３日目に１０容積％グルタルアルデヒドで室温２０分間固定した。対照として１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地のみで３日間培養し、細胞をグルタルアルデヒドで固定した。固定した細胞を、顕微鏡下で観察し、実験３の場合と同じ方法で神経突起伸展の有無を評価した。結果を表１６に併せて示す。なお、試験は、ＮＫ－５６及びＮＫ－５３を除く各試験試料溶液につき、トリプレットで２回実施し、その平均を求めた。また、この実験系にＮＧＦのみを添加（５ｎｇ／ｍｌ）したときの神経突起伸展率は５％程度であった。 <Evaluation of neurite outgrowth promoting action>
Similar to Evaluation B in Experiment 3, PC12-HS cells were diluted with 10% by volume FBS-added D-MEM medium to a collagen-coated 96-well microplate at 5 × 10 3 cells / well, and 100 μl / Seeded in wells. After 24 hours, 50 μl / well of each test sample solution adjusted to a compound concentration of 2 μg / ml and 50 μl / well of NGF (final concentration 5 ng / ml) were added, and 10% by volume glutaraldehyde was added at room temperature on the third day of culture. Fixed for 20 minutes. As a control, the cells were cultured for 3 days only in D-MEM medium supplemented with 10% by volume FBS, and the cells were fixed with glutaraldehyde. The fixed cells were observed under a microscope, and the presence or absence of neurite extension was evaluated by the same method as in Experiment 3. The results are also shown in Table 16. The test was performed twice with each triplet for each test sample solution except NK-56 and NK-53, and the average was obtained. Further, when only NGF was added to this experimental system (5 ng / ml), the neurite extension rate was about 5%.
Figure JPOXMLDOC01-appb-C000062
 　一般式３において、Ｒ７乃至Ｒ９は互いに同じか異なる脂肪族炭化水素基を表す。Ｘ３ －は適宜の対アニオンを表し、ｍはカチオン部の電荷とバランスする電荷となる１又は２のいずれかである整数を表す。
Figure JPOXMLDOC01-appb-C000062
 In the general formula 3, R 7 to R 9 represent the same or different aliphatic hydrocarbon groups. X 3 - represents an appropriate counter anion, m represents an integer of either charge become 1 or 2 to balance the charge of the cation.
Figure JPOXMLDOC01-appb-T000063
Figure JPOXMLDOC01-appb-T000063
　表１６から明らかなように、側鎖のアルキル基の炭素数が３乃至１２で、細胞増殖及び神経突起伸展の促進作用が認められ、３乃至１０でその作用は強くなり、細胞増殖促進作用は４乃至９のものが最も強い活性を示し、神経突起伸展作用は５乃至１０のものが最も強い活性を示した。また、ＮＫ－２４とＮＫ－５６、或いは、ＮＫ－１９とＮＫ－５３とを比較すると、何れの場合にも、神経突起伸展促進作用の程度に差は認められないことから、ＮＫ－１９類縁体の持つ神経栄養因子様活性には、対アニオンの種類による差異はないと判断した。 As is apparent from Table 16, when the side chain alkyl group has 3 to 12 carbon atoms, the cell growth and neurite outgrowth promoting effects are observed. Those with 4 to 9 showed the strongest activity, and those with neurite outgrowth of 5 to 10 showed the strongest activity. In addition, when NK-24 and NK-56, or NK-19 and NK-53 are compared, there is no difference in the degree of neurite outgrowth promoting action in either case. The neurotrophic factor-like activity of the body was judged to have no difference depending on the type of counter anion.
　以上の実験結果は、ＮＫ－１９、ＮＫ－５３、ＮＫ－１５０などの一般式３で表され、側鎖のアルキル基の炭素数が３乃至１０個の化合物、とりわけ、一般式６で表され、側鎖のアルキル基の炭素数が３乃至１０個の化合物は、神経栄養因子様作用、神経変性抑制作用などの生理機能を持っているので、これらの化合物はアルツハイマー病や小脳失調症などの抗神経変性疾患剤として有用であることを物語っている。 The above experimental results are represented by the general formula 3 such as NK-19, NK-53, NK-150, etc., and the compounds having 3 to 10 carbon atoms in the side chain alkyl group, especially represented by the general formula 6. Since compounds having 3 to 10 carbon atoms in the side chain alkyl group have physiological functions such as neurotrophic factor-like action and neurodegeneration inhibitory action, these compounds can be used for Alzheimer's disease, cerebellar ataxia, etc. It is useful as an anti-neurodegenerative disease agent.
＜実験１１：ＮＫ－４類縁体の神経栄養因子様活性＞
　実験１０と同様に、ＮＫ－４の類縁体に同様の作用効果があることを確認するために、下記一般式２で表される化合物において、側鎖のアルキル基（Ｒ４乃至Ｒ６）の炭素数が２乃至８で、対アニオンがＩ－である７種類の化合物を合成し、実験３と同様に、ＰＣ１２－ＨＳ細胞に対する細胞増殖促進作用、及び、神経突起伸展促進作用の強さを調べた。すなわち、ＮＫ－４に加えて、表１７に示すＮＫ－２３４、ＮＫ－２６、ＮＫ－９８１５、ＮＫ－９６９４、ＮＫ－２８及びＮＫ－１４７の７種類の化合物を、各々ＤＭＳＯに５ｍｇ／ｍｌとなるように溶解した。この溶液をそれぞれ化合物の終濃度が表１７又は１８に示す濃度となるように、１０容積％ＦＢＳ加Ｄ－ＭＥＭで希釈し試験試料溶液を調製した。また、ＮＫ－１９の類縁体ＮＫ－１３、ＮＫ－３９２、ＮＫ－１９及びＮＫ－１５０をＤＭＳＯに５ｍｇ／ｍｌとなるように溶解し、化合物の濃度が表１７又は１８に示す濃度となるように１０容積％ＦＢＳ加Ｄ－ＭＥＭで希釈し、試験試料溶液を調製した。なお、試験は、いずれも、各試験試料溶液につきトリプレットで２回実施し、その平均を求めた。細胞増殖促進作用の結果を表１７に、神経突起伸展促進作用の結果を表１８にそれぞれ併せて示す。 <Experiment 11: Neurotrophic factor-like activity of NK-4 analog>
As in Experiment 10, in order to confirm that the analog of NK-4 has the same effect, in the compound represented by the following general formula 2, the side chain alkyl group (R 4 to R 6 ) Seven types of compounds having 2 to 8 carbon atoms and a counter anion of I − were synthesized, and as in Experiment 3, the strength of cell proliferation promoting action and neurite extension promoting action on PC12-HS cells was increased. Examined. That is, in addition to NK-4, seven types of compounds shown in Table 17, NK-234, NK-26, NK-9815, NK-9694, NK-28 and NK-147, each in DMSO at 5 mg / ml It dissolved so that it might become. This solution was diluted with 10% by volume FBS-added D-MEM so that the final concentrations of the compounds were as shown in Table 17 or 18, respectively, to prepare test sample solutions. In addition, NK-19 analogs NK-13, NK-392, NK-19 and NK-150 are dissolved in DMSO to a concentration of 5 mg / ml, so that the concentration of the compound becomes the concentration shown in Table 17 or 18. Was diluted with 10% by volume FBS-added D-MEM to prepare a test sample solution. Each test was carried out twice with each triplet for each test sample solution, and the average was obtained. Table 17 shows the results of the cell growth promoting action, and Table 18 shows the results of the neurite extension promoting action.
Figure JPOXMLDOC01-appb-C000064
 　一般式２において、Ｒ４乃至Ｒ６は互いに同じか異なる脂肪族炭化水素基を表す。Ｘ２ －は適宜の対アニオンを表し、ｍはカチオン部の電荷とバランスする電荷となる１又は２のいずれかである整数を表す。
Figure JPOXMLDOC01-appb-C000064
 In General Formula 2, R 4 to R 6 represent the same or different aliphatic hydrocarbon groups. X 2 − represents an appropriate counter anion, and m represents an integer that is either 1 or 2 that has a charge balanced with the charge of the cation moiety.
Figure JPOXMLDOC01-appb-T000065
Figure JPOXMLDOC01-appb-T000065
Figure JPOXMLDOC01-appb-T000066
Figure JPOXMLDOC01-appb-T000066
　表１７及び１８の結果から明らかなように、一般式２で表されるＮＫ－４類縁体は、側鎖のアルキル基の炭素数が３乃至８化合物で、ＮＫ－４と同等乃至高い細胞増殖及び神経突起伸展の促進作用が認められ、化合物の濃度８０ｎｇ／ｍｌで比較すると、細胞増殖促進作用は側鎖のアルキル基の炭素数が４乃至６の化合物で特に強い作用が認められた。神経突起進展促進作用は側鎖のアルキル基の炭素数が４及び５の化合物で強い活性の発現が認められた。また、一般式３で表され、側鎖のアルキル基の炭素数が２乃至８である化合物、とりわけ、一般式３で表され、側鎖のアルキル基の炭素数が６乃至８であるＮＫ－１９類縁体化合物は強い細胞増殖及び神経突起伸展の促進活性を示すことが明らかになった。 As is clear from the results of Tables 17 and 18, the NK-4 analog represented by the general formula 2 has a compound having 3 to 8 carbon atoms in the side chain alkyl group, and has a cell growth equivalent to or higher than that of NK-4. In comparison with the compound concentration of 80 ng / ml, the cell growth promoting action was particularly strong for compounds having 4 to 6 carbon atoms in the side chain alkyl group. As for the neurite outgrowth promoting action, a strong activity was observed in the compounds having 4 and 5 carbon atoms in the side chain alkyl group. Further, a compound represented by the general formula 3 and having a side chain alkyl group having 2 to 8 carbon atoms, particularly, an NK— represented by the general formula 3 and having a side chain alkyl group having 6 to 8 carbon atoms. It was revealed that 19 analog compounds showed strong cell proliferation and neurite outgrowth promoting activity.
＜実験１２：６－ヒドロキシドーパによる細胞傷害に及ぼすＮＫ－４類縁体及びＮＫ－１９類縁体の影響＞
　ＮＫ－４類縁体及びＮＫ－１９類縁体化合物は強い細胞増殖及び神経突起伸展の促進活性を示すことが明らかになったので、本実験ではこれらの化合物の細胞傷害に及ぼす影響について調べた。すなわち、細胞傷害因子として６－ヒドロキシドーパを用いた。実験１と同様に１０容積％ＦＢＳ加Ｄ－ＭＥＭで培養したＰＣ１２－ＨＳ細胞を、９６ウエルマイクロプレートに２´１０４個／１００μｌ／ウエルとなるように播種した。２４時間後に１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈し、終濃度の２倍に調整した、ＮＫ－４、ＮＫ－９６９４、ＮＫ－１９及びＮＫ－１５０のいずれかを５０μｌと４００μＭ　６－ヒドロキシドーパ５０μｌとを添加し、３７℃、５容積％ＣＯ２ｉｎｃｕｂａｔｏｒ内で２４時間培養した後、１０％グルタルアルデヒドで固定し、定法に従いメチレンブルーを用いたダイアップテイク法により６５０ｎｍの吸光度を測定した。１０容積％ＦＢＳ加Ｄ－ＭＥＭを１００μｌ添加して２４時間培養した対照の細胞数（吸光度）を１００％としたときの相対値を求め各ウエルの細胞生存率（％）とした。結果を表１９に示す。 <Experiment 12: Effects of NK-4 Analogue and NK-19 Analogue on Cell Damage by 6-Hydroxydopa>
Since NK-4 analogs and NK-19 analog compounds have been shown to exhibit strong cell proliferation and neurite outgrowth promoting activity, the effects of these compounds on cytotoxicity were examined in this experiment. That is, 6-hydroxydopa was used as a cytotoxic factor. As in Experiment 1, PC12-HS cells cultured in 10% by volume FBS-added D-MEM were seeded in a 96-well microplate so that the concentration was 2'10 4 cells / 100 μl / well. After 24 hours, 50 μl and 400 μM 6-hydroxy were added to any one of NK-4, NK-9694, NK-19 and NK-150, diluted with 10% by volume FBS-added D-MEM medium and adjusted to twice the final concentration. After adding 50 μl of dopa and culturing at 37 ° C. in a 5 volume% CO 2 incubator for 24 hours, it was fixed with 10% glutaraldehyde, and the absorbance at 650 nm was measured by a die-up take method using methylene blue according to a conventional method. A relative value was determined when the number of cells (absorbance) of a control cultured for 24 hours after adding 100 μl of 10% by volume FBS-added D-MEM was taken as 100%, and was taken as the cell viability (%) of each well. The results are shown in Table 19.
Figure JPOXMLDOC01-appb-T000067
Figure JPOXMLDOC01-appb-T000067
　表１９から明らかなように、ＮＫ－４、ＮＫ－９６９４、ＮＫ－１９及びＮＫ－１５０は６－ヒドロキシドーパによる細胞傷害を有意に抑制した。 As is clear from Table 19, NK-4, NK-9694, NK-19, and NK-150 significantly inhibited cell damage caused by 6-hydroxydopa.
　６－ヒドロキシドーパは、パーキンソン病のｉｎ　ｖｉｖｏ　及び　ｉｎ　ｖｉｔｒｏ　モデルとして用いられるカテコールアミン作動性神経細胞選択的ニューロトキシンなので、この結果はＮＫ－４類縁体及びＮＫ－１９類縁体が、パーキンソン病の治療剤に利用できることを示唆している。また、この実験条件下では、市販されているエダラボンやドネペジルが６－ヒドロキシドーパによる細胞傷害を抑制しない１μｇ／ｍｌ以下の濃度で、これらの化合物は抑制作用を示すことから、ＮＫ－４、ＮＫ－９６９４、ＮＫ－１９及びＮＫ－１５０の方がエダラボンやドネペジルよりも６－ヒドロキシドーパによる細胞傷害を抑制する作用が強いと結論される。 6-Hydroxydopa is a catecholaminergic neuron-selective neurotoxin used as an in vivo and in vitro model of Parkinson's disease, and as a result, NK-4 analogs and NK-19 analogs are therapeutic agents for Parkinson's disease. It suggests that it can be used. Also, under these experimental conditions, commercially available edaravone or donepezil has an inhibitory action at a concentration of 1 μg / ml or less at which the cytotoxicity caused by 6-hydroxydopa is not inhibited. Therefore, NK-4, NK It is concluded that -9694, NK-19, and NK-150 are more effective in suppressing cell damage caused by 6-hydroxydopa than edaravone or donepezil.
　なお、具体的なデータは示さないが、ＰＣ－１２ＨＳ細胞に替えて、ラット胎児大脳皮質から調製した初代神経細胞（ニューロン、アストロサイト及びミクログリア細胞）を用い、アミロイドβフラグメント障害及び過酸化水素障害に対するＮＫ－４、ＮＫ－２６、ＮＫ－２３４、ＮＫ－１９及びＮＫ－１５０の影響を調べたところ、これらの化合物は初代神経細胞に対するアミロイドβフラグメント障害及び過酸化水素障害を抑制する活性を有することが判明した。また、これらの化合物は、ＬＰＳ存在下においてミクログリア細胞が産生するＮＯの産生を抑制することが判明した。 Although specific data are not shown, amyloid β fragment disorder and hydrogen peroxide disorder using primary neurons (neurons, astrocytes and microglia cells) prepared from rat fetal cerebral cortex instead of PC-12HS cells. NK-4, NK-26, NK-234, NK-19 and NK-150 were examined for their effects on these cells, and these compounds have activity to inhibit amyloid β fragment damage and hydrogen peroxide damage to primary neurons It has been found. In addition, these compounds were found to suppress the production of NO produced by microglia cells in the presence of LPS.
＜実験１３：ＮＫ－４類縁体又はＮＫ－１９類縁体の脳梗塞モデルラットに及ぼす影響＞
　ＰＣ－１２ＨＳ細胞に対する細胞増殖促進作用及び神経突起促進作用が確認されたＮＫ－４、ＮＫ－２６、ＮＫ－１５が脳梗塞に及ぼす影響を、ヒトの脳梗塞モデルラットを用いて調べた。すなわち、実験７に準じて、ＳＤラット（日本チャールスリバー社販売、オス、８週齢、体重２８０乃至３３０ｇ）に中大脳動脈に塞栓を行った。ＮＫ－１５０、ＮＫ－２６及びＮＫ－４を、それぞれＤＭＳＯに５ｍｇ／ｍｌの濃度で溶解後、ポワサイズ０．４５μｍの膜フィルターで膜濾過し、さらにＤＭＳＯで２．５乃至０．０５ｍｇ／ｍｌの濃度に調製したものを遮光し保存した。これらの化合物溶液を、使用時に生食で２５０倍希釈し、塞栓したラットに、閉塞１時間後および再開通時の２回、尾静脈より投与した（液量：５ｍｌ／ｋｇ・体重）。ＮＫ－４は１０ｍｇ／ｍｌの溶液を調製し、２５０乃至１６７倍希釈し、尾静脈より投与した（液量：５ｍｌ／ｋｇ・体重）。ＮＫ－４と同じ投与スケジュールで、陰性対照として生理食塩水を投与し、陽性対照として既存薬エダラボン（田辺三菱製薬社製、商品名「ラジカット」）をＤＭＳＯにより希釈し静脈内投与した。実験７と同じ方法により行動学的スコアを求め神経症状を評価した。また、脳梗塞部位の体積（ｍｍ３）は、梗塞に伴う脳の浮腫の影響を排除するため、予め腫脹率（＝虚血側大脳半球の体積÷健常側大脳半球の体積）を求め、梗塞体積の実測値を腫脹率で除して求めた。その結果と群構成を表２０に示した。 <Experiment 13: Effect of NK-4 analog or NK-19 analog on rat model of cerebral infarction>
The effects of NK-4, NK-26, and NK-15, which were confirmed to have cell growth promoting action and neurite promoting action on PC-12HS cells, on cerebral infarction were examined using human cerebral infarction model rats. That is, according to Experiment 7, SD rats (manufactured by Charles River Japan, male, 8 weeks old, body weight 280 to 330 g) were embolized in the middle cerebral artery. NK-150, NK-26, and NK-4 were each dissolved in DMSO at a concentration of 5 mg / ml, filtered through a membrane filter with a pore size of 0.45 μm, and further 2.5 to 0.05 mg / ml in DMSO. What was adjusted to the concentration was stored in the dark. These compound solutions were diluted 250-fold with saline at the time of use and administered to the embolized rat through the tail vein twice after 1 hour of occlusion and at the time of reopening (fluid amount: 5 ml / kg / body weight). NK-4 was prepared as a 10 mg / ml solution, diluted 250 to 167 times, and administered from the tail vein (fluid volume: 5 ml / kg / body weight). In the same administration schedule as NK-4, physiological saline was administered as a negative control, and an existing drug edaravone (manufactured by Mitsubishi Tanabe Pharma Corporation, trade name “Radicut”) was diluted with DMSO and administered intravenously as a positive control. A behavioral score was obtained by the same method as in Experiment 7 to evaluate neurological symptoms. Further, the volume of the cerebral infarction site (mm 3 ) is obtained in advance by determining the swelling rate (= volume of ischemic cerebral hemisphere ÷ volume of healthy cerebral hemisphere) in order to eliminate the influence of cerebral edema associated with infarction. The actual volume value was obtained by dividing by the swelling rate. The results and the group composition are shown in Table 20.
Figure JPOXMLDOC01-appb-T000068
Figure JPOXMLDOC01-appb-T000068
　表２０から明らかなように、ＮＫ－４、ＮＫ－２６及びＮＫ－１５０を投与したラットでは、至適投与量に差はあるものの、いずれも脳梗塞に伴う行動学的スコアの有意な改善と、脳梗塞部位の体積増加の有意な抑制が認められた。エダラボンの投与では行動学的スコアの改善は認められたものの、脳梗塞部位の体積増加の有意な抑制が認められなかった。この結果は、ＮＫ－４、ＮＫ－２６及びＮＫ－１５０は脳梗塞部位の体積増加を抑制し、脳梗塞に伴う運動障害を改善する作用を有することを物語っている。また、この実験系においては市販薬のエダラボンよりもＮＫ－４、ＮＫ－２６及びＮＫ－１５０の方が脳梗塞に伴う運動障害につき強い改善作用を示すことが判明した。 As is clear from Table 20, in rats administered with NK-4, NK-26, and NK-150, although there is a difference in the optimum dose, all of them showed a significant improvement in behavioral scores associated with cerebral infarction. In addition, a significant suppression of volume increase at the cerebral infarction site was observed. Administration of edaravone improved behavioral scores, but did not significantly suppress volume increase at the cerebral infarction site. This result indicates that NK-4, NK-26, and NK-150 have the effect of suppressing the volume increase at the cerebral infarction site and improving the movement disorder associated with cerebral infarction. Further, in this experimental system, it was found that NK-4, NK-26 and NK-150 have a stronger ameliorating effect on movement disorders associated with cerebral infarction than the commercial drug edaravone.
＜実験１４：ＮＫ－４類縁体のＡｃｈＥ活性阻害作用の比較＞
　既述のごとく、ＡｃｈＥ活性抑制剤はアルツハイマー型認知症治療剤として用いられている。そこで、ＮＫ－４類縁体をアルツハイマー症に適用することを想定しＮＫ－４類縁体のＡｃｈｅ活性阻害作用の強さを比較した。すなわち、実験１１で用いた一般式２で表する化合物の側鎖のアルキル基の炭素数が２乃至５の４種のＮＫ－４類縁体を用い、実験８と同じ方法でＡｃｈＥ活性残存率（％）を測定した。併せて、ＮＫ－１９類縁体のＮＫ－１５０を用いてＡｃｈｅ活性残存率（％）を測定した。その結果及斯かる活性残存率に基づき計算したＩＣ５０値（実験で用いたＡｃｈｅ活性を５０％抑制する化合物濃度）を表２１に併せて示す。 <Experiment 14: Comparison of AchE activity inhibitory action of NK-4 analog>
As described above, the AchE activity inhibitor is used as a therapeutic agent for Alzheimer-type dementia. Therefore, assuming that the NK-4 analog is applied to Alzheimer's disease, the strength of the Ache activity inhibitory action of the NK-4 analog was compared. That is, using the four NK-4 analogs having 2 to 5 carbon atoms in the side chain alkyl group of the compound represented by the general formula 2 used in Experiment 11, the remaining AchE activity ( %). In addition, the remaining percentage of Ache activity (%) was measured using NK-19 analog NK-150. Table 21 also shows the IC50 values (compound concentrations that inhibit the Ache activity used in the experiment by 50%) calculated based on the results and the residual activity rate.
Figure JPOXMLDOC01-appb-T000069
Figure JPOXMLDOC01-appb-T000069
　表２１から明らかなように、ＮＫ－４類縁体は側鎖のアルキル基の炭素数が増えるにつれてＡｃｈｅ活性の残存率が高くなる傾向を示した。ＮＫ－４及びＮＫ－２３４、とりわけＮＫ－４はＡｃｈＥ活性を強く抑制した。また、ＮＫ－１９類縁体のＮＫ－１５０は、ＮＫ－４類縁体と比較しＡｃｈｅ抑制作用が低いことが判明した。 As is apparent from Table 21, the NK-4 analogs showed a tendency that the residual ratio of the Ache activity increased as the number of carbon atoms of the side chain alkyl group increased. NK-4 and NK-234, especially NK-4, strongly suppressed AchE activity. It was also found that NK-19 analog NK-150 has a lower Ache inhibitory effect than NK-4 analog.
　この結果は、ＮＫ－４及びＮＫ－２３４、とりわけＮＫ－４はアルツハイマー型認知症治療剤として有用であることを物語っている。また、Ａｃｈｅ活性抑制剤としてアルツハイマー型認知症治療用に市販されているドネペジル塩酸塩（商品名『アリセプト』）のＡｃｈｅ活性残存率（％）を同じ実験系で測定したところＩＣ５０は０．９μｇ／ｍｌとなったことから、ＮＫ－４はドネペジル塩酸塩とほぼ同等のＡｃｈｅ活性抑制活性を有することを物語っている。 This result indicates that NK-4 and NK-234, especially NK-4, is useful as a therapeutic agent for Alzheimer-type dementia. Further, when the Ache activity remaining rate (%) of donepezil hydrochloride (trade name “Aricept”) marketed for the treatment of Alzheimer-type dementia as an Ache activity inhibitor was measured in the same experimental system, the IC 50 was 0.9 μg. / Ml, NK-4 has almost the same activity of inhibiting Ache activity as donepezil hydrochloride.
＜実験１５：ＮＫ－４類縁体及びＮＫ－１９類縁体のヒトアルツハイマー型認知症のモデルマウスに及ぼす影響＞
　既述の実験からＮＫ－４がアルツハイマー型認知症治療剤として利用できる可能性が示唆されたので、本実験ではＮＫ－４類縁体及びＮＫ－１９類縁体のヒトアルツハイマー型認知症のモデルマウスに及ぼす影響を検証した。 <Experiment 15: Effects of NK-4 analog and NK-19 analog on model mice of human Alzheimer type dementia>
The above experiments suggested that NK-4 can be used as a therapeutic agent for Alzheimer's dementia. In this experiment, NK-4 analogs and NK-19 analogs were modeled on human Alzheimer's model mice. The effect was verified.
＜被験試料＞
　被験試料としてＮＫ－４、ＮＫ－２３４、ＮＫ－２６、ＮＫ－１９及びＮＫ－１５０を用いた。対照１として生理食塩水（２００μｌ／匹）を投与した。対照２としてドネペジル塩酸塩を用いた。各被験試料はＤＭＳＯにより５ｍｇ／ｍｌの濃度に溶解し、生理食塩水に希釈し投与した。 <Test sample>
NK-4, NK-234, NK-26, NK-19 and NK-150 were used as test samples. As a control 1, physiological saline (200 μl / animal) was administered. Donepezil hydrochloride was used as control 2. Each test sample was dissolved in DMSO to a concentration of 5 mg / ml, and diluted with physiological saline for administration.
＜実験方法＞
　ＩＣＲマウス（日本チャールスリバー社販売、オス、５週齢、体重２５乃至３０ｇ）１００匹を無作為に１０群各１０匹に群分けし、試験終了まで単独飼育した。抱水クロラール（シグマ社販売、３５０ｍｇ／ｋｇ・体重、腹腔内投与）麻酔を施したマウスを、背面固定し、頭部正中切開を加え、骨縫合を確認した後、ブレグマの左側方１．０ｍｍ、後方０．５ｍｍに刺入点を定め、３ｍｍ刺入し、配列表における配列番号１で示すアミノ酸配列を有するアミロイドβフラグメント（β－Ａｍｙｌｏｉｄ２５－３５）溶液９ｎｍｏｌ／６μｌ／匹を脳室内に投与した（投与法は、『Ｂｒａｉｎ　Ｒｅｓｅａｒｃｈ』、第７０６巻、１８１－１９３頁（１９９６年）参照。）。投与には２８ゲージステンレス針（３ｍｍ）を装着したマイクロシリンジを用いた。刺入部位は、予めアミロイドβフラグメント溶液の代わりにエバンスブルー溶液（０．３μｇ／０．３μｌ）を投与し、左右前額断面の側脳室、背側第三脳室、腹側第三脳室などに着色が認められることを確認し決定した。投与後、頭皮を縫合し、翌日より化合物のいずれかを腹腔内に１日１回１３日間投与し、以下に示す方法により行動学的評価をおこなった。その結果と群構成を表２２に示した。 <Experiment method>
100 ICR mice (manufactured by Charles River Japan, male, 5 weeks old, body weight 25 to 30 g) were randomly divided into 10 groups of 10 mice and reared alone until the end of the test. Chloral hydrate (SIGMA, 350 mg / kg, body weight, intraperitoneal administration) Anesthetized mice were fixed on the back, and a midline incision was made on the head. After confirming bone suture, 1.0 mm on the left side of Bregma The insertion point was determined 0.5 mm backward, 3 mm insertion was performed, and 9 nmol / 6 μl / animal solution of amyloid β fragment (β-Amyloid 25-35 ) having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing was placed in the ventricle (Refer to “Brain Research”, 706, 181-193 (1996) for the administration method.) For administration, a microsyringe equipped with a 28 gauge stainless needle (3 mm) was used. Evans blue solution (0.3 μg / 0.3 μl) is administered in advance for the insertion site instead of the amyloid β fragment solution, and the lateral ventricle, dorsal third ventricle, ventral third brain of the left and right forehead cross section It was confirmed that coloring was observed in the room. After the administration, the scalp was sutured, and one of the compounds was intraperitoneally administered once a day for 13 days from the next day, and behavioral evaluation was performed by the following method. The results and group composition are shown in Table 22.
Figure JPOXMLDOC01-appb-T000070
Figure JPOXMLDOC01-appb-T000070
＜評価法＞ <Evaluation method>
＜新奇物体認識試験＞
　新奇物体認識試験は、マウスの新奇性を好むとい特性を利用したもので、他の多くの学習評価系と異なり人為的な強化因子を用いない。試験は、順化、訓練試行、保持試行の３部門で構成され、アミロイドβフラグメントを脳室内に投与して後６乃至８日目に実施した。床にウッドチップを敷き詰めたオープンフィールドの実験装置（縦４０ｃｍ、横３０ｃｍ、高さ３０ｃｍ）を、約１，０００ルックス（ｌｕｘ）の照明下、雑音のない場所に設置した。まず６日目に、マウスを探索物体の入っていない実験装置中央に入れ、１０分間自由に探索させた（順化）。その２４時間後（７日目）、実験装置内に２種類の物体（ＡとＢ）をそれぞれ側面から１０ｃｍの位置に設置し、マウスを実験装置中央に入れ、１０分間自由に探索させた（訓練試行）。さらにその２４時間後（８日目）、実験装置内に前日探索させた物体Ａを前日の物体Ａ（一度記憶した対象物）と同じ位置に、前日の物体Ｂと異なる物体Ｃ（新しい対象物）を物体Ｂと同じ位置に設置し、マウスを装置の中央に入れ１０分間自由に探索させた（保持試行）。この時、マウスが鼻先を物体に向け、鼻先から物体までの距離が２ｃｍ以内にある時、又はマウスの鼻先が物体に接触している時を物体探索中とみなし、その時間をストップウオッチで計測した。物体識別指数（＝（新しい対象物の探索に費やした時間－一度記憶した対象物の探索に費やした時間）／（新しい対象物の探索に費やした時間＋一度記憶した対象物の探索に費やした時間））を求めた。この場合、物体識別指数は、新規対象物の探索により多く割かれた時間の全探索時間に対する割合であり、一度探索した対象物を動物が記憶していれば物体識別指数の値が大きくなり、記憶していなければ値が小さくなる。 <New object recognition test>
The novel object recognition test uses the characteristics of the mouse when it likes the novelty, and unlike many other learning evaluation systems, it does not use artificial reinforcement factors. The test consisted of three departments: acclimatization, training trials, and retention trials, which were performed 6-8 days after amyloid β fragment was administered into the ventricle. An open field experimental device (40 cm long, 30 cm wide, 30 cm high) with wood chips on the floor was installed in a noise-free place under about 1,000 lux lighting. First, on the 6th day, the mouse was placed in the center of the experimental apparatus without the search object and allowed to search freely for 10 minutes (acclimation). Twenty-four hours later (7th day), two types of objects (A and B) were placed in the experimental apparatus at positions 10 cm from the side, and the mouse was placed in the center of the experimental apparatus and allowed to search freely for 10 minutes ( Training trial). Furthermore, 24 hours later (8th day), the object A searched for in the experiment device the previous day is located at the same position as the object A (the object stored once) on the previous day, and the object C (new object) different from the object B on the previous day. ) Was placed at the same position as the object B, and the mouse was placed in the center of the apparatus and allowed to search freely for 10 minutes (holding trial). At this time, when the mouse pointed at the tip of the nose and the distance from the tip of the nose to the object was within 2 cm, or when the nose of the mouse was in contact with the object, the object search was considered and the time was measured with a stopwatch. . Object identification index (= (time spent searching for a new object−time spent searching for an object once stored) / (time spent searching for a new object + searching for an object stored once) Time)). In this case, the object identification index is the ratio of the time allotted to the search for new objects to the total search time, and if the animal stores the object once searched, the value of the object identification index becomes large, If not stored, the value becomes smaller.
＜受動的回避試験＞
　動物が一度経験した嫌悪刺激（電気刺激）に対して示す回避行動を記憶の指標とするもので、マウスが暗室を好む性質を利用したステップスルー型を採用した。明室と暗室が扉でつながった装置の明室側にマウスを入れた時の暗室側への移動時間を記憶の指標とした。受動的回避試験はアミロイドβフラグメントを脳室内に投与して後９乃至１２日目に実施した。９日目に明室（１，０００ルクス、縦３０ｃｍ、横３０ｃｍ、高さ１５ｃｍ）に１分間、暗室（縦３０ｃｍ、横３０ｃｍ、高さ１５ｃｍ）に２分間入れ順化させた。１０日目も同様に順化を行った。１１日目の訓練試行で、まず明室の中央にマウスを入れ、マウスが暗室内に移動すると同時に明室と暗室のとの間の扉を閉め、電気刺激を与えた（０．８ｍＡ、１秒）。２４時間後（１２日目）、前日と同様に再び明室の中央にマウスを入れ、暗室への移動時間（秒）を受動的回避的反応として測定した。通電による嫌悪刺激を記憶していれば受動的回避的反応が長くなる。 <Passive avoidance test>
A step-through type using the property that the mouse prefers the dark room is adopted as an index of memory, which is the avoidance behavior shown to the aversive stimulation (electrical stimulation) once experienced by the animal. The movement time to the dark room side when the mouse was put in the light room side of the device where the light room and dark room were connected by a door was used as an index of memory. The passive avoidance test was performed 9 to 12 days after amyloid β fragment was administered into the ventricle. On the 9th day, the light room (1,000 lux, length 30 cm, width 30 cm, height 15 cm) was placed in a dark room (length 30 cm, width 30 cm, height 15 cm) for 2 minutes to acclimatize. The acclimatization was similarly performed on the 10th day. In the training trial on the 11th day, the mouse was first placed in the center of the light room, and at the same time the mouse moved into the dark room, the door between the light room and the dark room was closed, and electrical stimulation was applied (0.8 mA, 1 Seconds). After 24 hours (day 12), the mouse was placed in the center of the light room again as in the previous day, and the moving time (seconds) to the dark room was measured as a passive avoidance response. If the aversive stimulus by energization is memorized, the passive avoidance reaction becomes longer.
　表２２から明らかなように、ＮＫ－４を５００μｇ／ｋｇ・体重、ＮＫ－２３４を５００μｇ／ｋｇ・体重、ＮＫ－２６を５０μｇ／ｋｇ・体重、ＮＫ－１９を５００μｇ／ｋｇ・体重及びＮＫ－１５０を５００μｇ／ｋｇ・体重投与マウスでは、アミロイドβフラグメントのみ投与マウスに比べて、新奇物体認識試験で有意の改善が認められた。また、ＮＫ－４を５０μｇ／ｋｇ・体重、ＮＫ－４を５００μｇ／ｋｇ・体重、ＮＫ－２３４を５００μｇ／ｋｇ・体重、ＮＫ－２６を５０μｇ／ｋｇ・体重、ＮＫ－２６を５００μｇ／ｋｇ・体重、及び、ＮＫ－１９を５００μｇ／ｋｇ・体重投与マウスでは、アミロイドβフラグメントのみを投与マウスに比べて、受動的回避試験で有意の改善が認められた。なかでも、ＮＫ－４を５００μｇ／ｋｇ・体重投与した群では、新奇物体認識試験及び受動的回避試験の両方で、ドネペジル塩酸塩１，０００μｇ／ｋｇ・体重投与した群（対照２）よりも著明な改善が認められた。なお、試験中ＮＫ－４、ＮＫ－２３４、ＮＫ－２６、ＮＫ－１９或いはＮＫ－１５０の投与に起因すると思われる副作用は認められなかった。 As is apparent from Table 22, NK-4 is 500 μg / kg · body weight, NK-234 is 500 μg / kg · body weight, NK-26 is 50 μg / kg · body weight, NK-19 is 500 μg / kg · body weight and NK− In mice administered with 150 at 500 μg / kg · body weight, a significant improvement was observed in the novel object recognition test compared to mice administered with amyloid β fragment alone. Also, NK-4 is 50 μg / kg · body weight, NK-4 is 500 μg / kg · body weight, NK-234 is 500 μg / kg · body weight, NK-26 is 50 μg / kg · body weight, NK-26 is 500 μg / kg · body weight. Significant improvement was observed in the passive avoidance test in mice administered with body weight and NK-19 at 500 μg / kg / body weight, compared with mice administered with amyloid β fragment alone. In particular, in the group administered NK-4 at 500 μg / kg / body weight, both the novel object recognition test and the passive avoidance test were more marked than the group treated with donepezil hydrochloride at 1,000 μg / kg / body weight (control 2). A clear improvement was observed. During the study, no side effects believed to be caused by administration of NK-4, NK-234, NK-26, NK-19 or NK-150 were observed.
　この結果は、ＮＫ－２３４、ＮＫ－２６、ＮＫ－１９及びＮＫ－１５０は、いずれもアミロイドβペプチドに起因する認知障害を改善する作用を有し、この実験条件において、試験に用いた４種類の化合物の中ではＮＫ－４が最も強い認知障害改善作用を有していることを物語っている。 As a result, NK-234, NK-26, NK-19, and NK-150 all have an action to improve cognitive impairment caused by amyloid β peptide. Among these compounds, NK-4 has the strongest cognitive impairment improving effect.
＜実験１６：ＮＫ－４のＡＰＰトランスジェニックマウスに及ぼす影響＞
　実験１４で、アミロイドβフラグメント投与したマウスの認知障害改善作用の最も強かったＮＫ－４につき、スウェーデン型アルツハイマー病の原因遺伝子変異を導入した市販のＡＰＰトランスジェニックマウス（ＡＰＰ　Ｔｇマウス）に及ぼす影響を調べた。すなわち、ＡＰＰ　Ｔｇマウス（Ｔａｃｏｎｉｃ社販売、メス、１０週齢、体重１５乃至２３ｇ）４５匹を１０日間予備飼育した後、体重が均等になるように、４群に分け、単独飼育とし、ＮＫ－４を腹腔内に週５回、１２週間投与した。対照１として遺伝子変異を導入していないマウス（野生型、メス、１０週齢、体重１５乃至２３ｇ）１０匹を１０日間予備飼育後、単独飼育とし、生理食塩水を腹腔内に週５回、１２週間投与した。対照２としてＡＰＰ　Ｔｇマウスに生理食塩水（２００μｌ／匹）を週５回、１２週間投与した。対照３としてＡＰＰ　Ｔｇマウスにドネペジル塩酸塩を週５回、１２週間投与した。ＮＫ－４、生理食塩水或いはドネペジル塩酸塩投与１２週目に、実験１５と同じ方法で、最初に新奇物体認識試験、次に受動的回避試験を行い、引き続き下記方法による水迷路試験を行った。その結果と群構成を表２３に示す。 <Experiment 16: Effect of NK-4 on APP transgenic mice>
In Experiment 14, the effect of NK-4, which had the strongest effect on improving cognitive impairment in mice treated with amyloid β fragment, on commercially available APP transgenic mice (APP Tg mice) into which a causative gene mutation of Swedish Alzheimer's disease was introduced. Examined. Specifically, 45 APP Tg mice (taconic, female, 10 weeks old, body weight 15 to 23 g) were preliminarily reared for 10 days, then divided into 4 groups so that the body weights were equalized, and were reared alone, and NK- 4 was administered intraperitoneally 5 times a week for 12 weeks. As a control 1, 10 mice (wild type, female, 10 weeks old, body weight 15 to 23 g) that were not introduced with a genetic mutation were preliminarily raised for 10 days, and then alone, and physiological saline was intraperitoneally injected 5 times a week. Administered for 12 weeks. As control 2, physiological saline (200 μl / mouse) was administered to APP Tg mice 5 times a week for 12 weeks. As a control 3, APP Tg mice were administered donepezil hydrochloride 5 times a week for 12 weeks. At the 12th week after administration of NK-4, physiological saline or donepezil hydrochloride, a novel object recognition test and then a passive avoidance test were first conducted in the same manner as in Experiment 15, followed by a water maze test by the following method. . The results and group composition are shown in Table 23.
Figure JPOXMLDOC01-appb-T000071
Figure JPOXMLDOC01-appb-T000071
＜水迷路試験法＞
　直径１３０ｃｍの円形プールに、白色インクで着色した水を深さ２０ｃｍまで満たし、水槽用ヒーターで水温を２３±１℃に維持した。プールを４分割し一画の中央に、プールの側面から１０ｃｍの位置に避難用のプラットホームを水面下２ｃｍになるように設置した。このプラットホームの位置は、試験終了まで一定の場所とした。受動的回避試験終了の翌日よりマウスをプールの側面に向けて水面上に放ち、水面下に隠れたプラットホームに到着するまでの時間を計測した。スタート位置は、プールを４分割したいずれかの画分の中央部、壁面より１０ｃｍ離れた場所とし、試行毎にランダムに変更した。２分間自由にプラットホームを探索させた後、マウスが２分以内にプラットホームに到着できなかった場合は、プラットホームへ誘導し、３０秒間プラットホームに留まらせた後、ペーパータオルを敷いたケージに移した。２回目の試験は、１回目の試験終了１分後に開始した。この試験を４日間連続で行ない、２回の試行の平均値を１日の値とした。 <Water maze test method>
A circular pool with a diameter of 130 cm was filled with water colored with white ink to a depth of 20 cm, and the water temperature was maintained at 23 ± 1 ° C. with a water tank heater. The pool was divided into four parts, and an evacuation platform was installed at a position 10 cm from the side of the pool so as to be 2 cm below the surface of the water. The platform position was fixed until the end of the test. From the day after the end of the passive avoidance test, the mouse was released on the surface of the pool toward the side of the pool, and the time taken to reach the platform hidden under the surface of the water was measured. The starting position was 10 cm away from the central part of one of the fractions of the pool divided into 4 centimeters, and was randomly changed for each trial. After searching the platform freely for 2 minutes, if the mouse could not reach the platform within 2 minutes, it was guided to the platform, allowed to stay on the platform for 30 seconds and then transferred to a cage with paper towels. The second test started 1 minute after the end of the first test. This test was conducted for 4 consecutive days, and the average value of the two trials was taken as the value of the day.
　表２３から明らかなように、ＮＫ－４を投与したマウスは、物体識別指数、受動的回避反応、水迷路試験の何れにおいても、ＡＰＰ　Ｔｇマウスの認知障害を顕著に改善した。また、その改善作用は、市販のアルツハイマー型認知症治療剤ドネペジル塩酸塩より有意に強かった。この結果は、ＮＫ－４がアルツハイマー型認知障害の治療剤として利用できることを強く示唆している。なお、試験中ＮＫ－４の投与に起因すると思われる副作用は認められなかった。 As is clear from Table 23, mice administered NK-4 markedly improved the cognitive impairment of APP Tg mice in any of the object identification index, passive avoidance reaction, and water maze test. Moreover, the improvement effect was significantly stronger than that of commercially available Alzheimer-type dementia therapeutic agent donepezil hydrochloride. This result strongly suggests that NK-4 can be used as a therapeutic agent for Alzheimer-type cognitive impairment. During the study, no side effects believed to be caused by administration of NK-4 were observed.
＜実験１６：脳血管性認知症モデルマウスに及ぼすＮＫ－４の影響＞
　既述の実験により、ＮＫ－４が脳梗塞に由来する運動障害やアルツハイマー型認知障害の改善に有効であることが確認できたので、本実験では血管性認知障害に対するＮＫ－４の影響を調べた。すなわち、Ｃ５７ＢＬ／６Ｊマウス（日本クレア社販売、雄、１２週齢）３１匹を１週間予備飼育後、２１匹にアトロピン（０．３ｍｇ／ｋｇ、皮下投与）を前投与した後、ペントバルビタールナトリウム（５０ｍｇ／ｋｇ）を腹腔内投与し麻酔し右総頸動脈の永久結紮手術を行った（手術法については特開２００８－１９３９４１号公報参照）。手術して後全てのマウスを単独飼育とし、自由飲食、飲水で飼育した。結紮手術を施した２１匹のうち１０匹はそのまま飼育（結紮群）し、残りの１１匹はＮＫ－４を投与した（投与群）。また手術を行っていない１０匹を対照（無手術群）とした。手術後２日目より、無手術群および結紮群には生理食塩水を、ＮＫ－４投与群にはＮＫ－４（１００μｇ／ｋｇ）を連日（週５日間）腹腔内投与した。手術３週目、４週目に、実験１５と同じ方法により新奇物体認識試験を実施した。その結果を表２４に示す。 <Experiment 16: Effect of NK-4 on cerebrovascular dementia model mice>
The above-mentioned experiment confirmed that NK-4 is effective in improving dyskinesia and Alzheimer-type cognitive impairment due to cerebral infarction. It was. Specifically, 31 C57BL / 6J mice (manufactured by Claire Japan, male, 12 weeks old) were preliminarily raised for 1 week, and then atropine (0.3 mg / kg, subcutaneous administration) was pre-administered to 21 mice, followed by pentobarbital sodium. (50 mg / kg) was intraperitoneally administered and anesthetized, and a permanent ligation operation was performed on the right common carotid artery (refer to Japanese Patent Application Laid-Open No. 2008-193941 for the operation method). After the operation, all mice were reared alone, and were bred with free eating and drinking water. Of the 21 animals subjected to ligation surgery, 10 animals were reared as they were (ligation group), and the remaining 11 animals were administered NK-4 (administration group). In addition, 10 animals that had not undergone surgery were used as controls (no surgery group). From the second day after the operation, physiological saline was administered intraperitoneally to the non-operative group and ligation group, and NK-4 (100 μg / kg) was intraperitoneally administered daily (5 days a week) to the NK-4 administration group. A novel object recognition test was performed in the same manner as in Experiment 15 on the third and fourth weeks of the operation. The results are shown in Table 24.
Figure JPOXMLDOC01-appb-T000072
Figure JPOXMLDOC01-appb-T000072
　表２４から明らかなように、ＮＫ－４投与群の物体識別指数は、無手術群と差が無く、右総頸動脈結紮による脳血管性認知障害をほぼ完全に回復させた。この結果は、ＮＫ－４が血管性認知障害の治療剤として利用できることを強く示唆している。なお、試験中ＮＫ－４の投与に起因すると思われる副作用は認められなかった。 As is apparent from Table 24, the object identification index of the NK-4 administration group was not different from the non-operative group, and cerebrovascular cognitive impairment due to right common carotid artery ligation was almost completely recovered. This result strongly suggests that NK-4 can be used as a therapeutic agent for vascular cognitive impairment. During the study, no side effects believed to be caused by administration of NK-4 were observed.
　以上の実験結果は、ＮＫ－１９、ＮＫ－５３、ＮＫ－１５０などの一般式３で表され、側鎖のアルキル基の炭素数が３乃至１０の化合物と同様に、一般式２で表され、側鎖のアルキル基の炭素数が２乃至８である化合物は、神経変性抑制作用を持っているので、これらの化合物はいずれも、脳梗塞による認知障害、運動障害、アルツハイマー型認知障害、血管性認知障害、小脳失調症などの治療用の抗神経変性症疾患剤として有用であることを物語っている。なかでも、一般式２で表され側鎖のアルキル基の炭素数が２乃至４である化合物であるＮＫ－４、ＮＫ－２３４、ＮＫ－２６は、神経変性抑制作用が強く、特にＮＫ－４は、脳梗塞による認知障害、運動障害、アルツハイマー型認知障害、血管性認知障害、パーキンソ氏病などの神経変性に伴う諸症状の改善作用に優れていると結論される。 The above experimental results are represented by the general formula 3 such as NK-19, NK-53, NK-150, etc., and are represented by the general formula 2 like the compounds having 3 to 10 carbon atoms in the side chain alkyl group. Since compounds having 2 to 8 carbon atoms in the side chain alkyl group have an inhibitory effect on neurodegeneration, all of these compounds have cognitive impairment due to cerebral infarction, motor impairment, Alzheimer type cognitive impairment, blood vessel It is useful as an anti-neurodegenerative disease agent for the treatment of sexual cognitive impairment and cerebellar ataxia. Among them, NK-4, NK-234, and NK-26, which are compounds represented by the general formula 2 and have 2 to 4 carbon atoms in the side chain alkyl group, have a strong neurodegeneration inhibitory effect, and particularly NK-4. It is concluded that it is excellent in improving various symptoms associated with neurodegeneration such as cognitive impairment due to cerebral infarction, motor impairment, Alzheimer type cognitive impairment, vascular cognitive impairment, and Parkinson's disease.
　以下、本発明の抗神経変性疾患剤について、実施例により説明するが、本発明はこれら実施例に何ら限定されるものではない。 Hereinafter, the anti-neurodegenerative disease agent of the present invention will be described with reference to examples, but the present invention is not limited to these examples.
＜注射用の液剤＞
　注射用精製水３７０ｇに注射用精製マルトース（株式会社林原製造）６０ｇを溶解した溶液と、注射用精製水１７０ｇに、有効成分として、ＮＫ－４（化学式２で表される化合物）、ＮＫ－２６（化学式１で表される化合物）、ＮＫ－２８（一般式２で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が７である化合物）、ＮＫ－１４７（一般式２で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が８である化合物）、ＮＫ－１９（化学式４で表される化合物）、ＮＫ－５３（化学式５で表される化合物）、ＮＫ－１５０（化学式３で表される化合物）、ＮＫ－３９３（一般式３で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が８である化合物）、ＮＫ－１００（化学式６で表される化合物）ＮＫ－５２８（化学式７で表される化合物）、ＮＫ－５５７（化学式８で表される化合物）、及び、ＮＫ－１５１６（化学式９で表される化合物）（いずれも株式会社林原生物化学研究所製造）のいずれか１種を、各々１２ｍｇ溶解した溶液とを混合し、濾過滅菌後、溶存する酸素の濃度が約０．１ｐｐｍになるまで無菌の窒素ガスをバブリングして、褐色アンプルに１ｍｌずつ分注し、窒素気流下でアンプルを封止した。本品は、いずれもパイロジェンフリーであり、抗神経変性疾患剤として利用できる。また、本品は、神経変性抑制剤、神経細胞保護剤、神経突起促進剤や、神経変性に伴う病態や神経機能障害の治療剤としても利用できる。また、本品は脳保護剤、脳の酸化的障害抑制剤、虚血性脳障害抑制剤、脳梗塞巣進展抑制剤、脳浮腫抑制剤、遅発性神経死抑制剤、脳機能正常化剤、酸化ストレス抑制剤、抗潰瘍剤、血糖上昇抑制剤、眼性疾患の予防・治療剤、移植臓器保存剤、移植組織・臓器の壊死防止剤、組織・臓器の障害の予防・治療剤、放射線障害予防・治療剤、抗腫瘍剤、腫瘍転移抑制剤、細胞障害マーカー抑制剤、炎症性疾患やそれに伴う組織障害の予防・治療剤、感覚細胞、感覚神経或いは感覚器の障害の抑制剤、薬物中毒の予防・治療剤、カルシウム・ナトリウム交換系阻害剤、疼痛や掻痒の予防・治療剤、プロテインキナーゼ刺激剤、ミトコンドリア脳筋症予防・治療剤、動脈閉塞・狭窄予防・治療剤、血液脳関門破綻抑制剤、薬物依存症治療剤、アポトーシス抑制剤、過酸化脂質生成抑制剤、ラジカルスカベンジャー、アミロイドβペプチド凝集阻害剤、アミロイドβペプチド傷害抑制剤、アセチルコリンエステラーゼ（ＡｃｈＥ）活性阻害剤、セリン／スレオニンキナーゼ（Ａｋｔ）活性化剤、ホスファチヂルイノシトール（３，４，５）３リン酸キナーゼ（ＰＩ３Ｋ）－セリン／スレオニンキナーゼ（Ａｋｔ）カスケード活性化剤、サイクリックＡＭＰ濃度上昇促進剤、或いは、ＳＡＰＫ／ＪＮＫリン酸化抑制剤として利用してもよい。さらに、本発明の抗神経変性疾患剤は、神経変性疾患を発症したペットをはじめとするヒト以外の動物の治療剤や、その予防剤として使用することもできる。 <Liquid preparation for injection>
A solution prepared by dissolving 60 g of purified maltose for injection (manufactured by Hayashibara Co., Ltd.) in 370 g of purified water for injection, NK-4 (compound represented by Chemical Formula 2), NK-26 as an active ingredient in 170 g of purified water for injection (Compound represented by chemical formula 1), NK-28 (compound in which the carbon number of the alkyl group (R) in the side chain of the compound represented by general formula 2 is 7), NK-147 (expressed by general formula 2) A compound in which the alkyl group (R) in the side chain of the compound has 8 carbon atoms), NK-19 (a compound represented by Chemical Formula 4), NK-53 (a compound represented by Chemical Formula 5), NK- 150 (compound represented by chemical formula 3), NK-393 (compound in which the side chain alkyl group (R) of the compound represented by general formula 3 has 8 carbon atoms), NK-100 (compound represented by chemical formula 6) Compound) NK-528 (Represented by Chemical Formula 7) Compound), NK-557 (compound represented by chemical formula 8), and NK-1516 (compound represented by chemical formula 9) (all manufactured by Hayashibara Biochemical Laboratories, Inc.) Mix each 12mg dissolved solution, filter sterilize, bubbling sterile nitrogen gas until dissolved oxygen concentration is about 0.1ppm, dispense 1ml each into brown ampoules, and ampoules under nitrogen stream Was sealed. All of this product is pyrogen-free and can be used as an anti-neurodegenerative disease agent. In addition, the product can be used as a neurodegeneration inhibitor, a nerve cell protective agent, a neurite promoter, or a therapeutic agent for a disease state or neurological dysfunction associated with neurodegeneration. In addition, this product is a brain protective agent, brain oxidative disorder inhibitor, ischemic brain disorder inhibitor, cerebral infarction growth inhibitor, brain edema inhibitor, delayed neuronal death inhibitor, brain function normalizing agent, Oxidative stress inhibitor, anti-ulcer agent, blood sugar elevation inhibitor, prevention / treatment agent for ocular diseases, transplanted organ preservative, transplanted tissue / organ necrosis inhibitor, tissue / organ injury preventive / therapeutic agent, radiation damage Prophylactic / therapeutic agent, antitumor agent, tumor metastasis inhibitor, cytopathic marker inhibitor, prophylactic / therapeutic agent for inflammatory diseases and associated tissue disorders, sensory cell, sensory nerve or sensory organ disorder inhibitor, drug addiction Preventive / therapeutic agent, calcium / sodium exchange inhibitor, preventive / therapeutic agent for pain and pruritus, protein kinase stimulator, preventive / therapeutic agent for mitochondrial encephalomyopathy, preventive / therapeutic agent for arterial occlusion / stenosis, blood-brain barrier disruption Inhibitors, drug dependence treatments, Apot Cis inhibitor, lipid peroxide production inhibitor, radical scavenger, amyloid β peptide aggregation inhibitor, amyloid β peptide injury inhibitor, acetylcholinesterase (AchE) activity inhibitor, serine / threonine kinase (Akt) activator, phosphati Diluinositol (3,4,5) 3-phosphate kinase (PI3K)-Serine / threonine kinase (Akt) cascade activator, cyclic AMP concentration increase promoter, or SAPK / JNK phosphorylation inhibitor Also good. Furthermore, the anti-neurodegenerative disease agent of the present invention can also be used as a therapeutic agent for a non-human animal including a pet that has developed a neurodegenerative disease, or as a preventive agent thereof.
　これらの製剤を使用して、小脳失調症ハムスターの運動協調性低下に対する効果及びアミロイドβフラグメント投与マウス（アルツハイマー病モデル）に対する治療効果を確認した。 Using these preparations, the effect of cerebellar ataxia hamsters on motor coordination decline and the therapeutic effect on amyloid β fragment-administered mice (Alzheimer's disease model) were confirmed.
＜小脳失調症ハムスターの運動協調性低下に対する効果＞
　実験２と同様に、同一週に生まれた３週齢の小脳失調症ハムスター１３０匹を、無作為に、１３群各１０匹に分けた。そのうちの１２群各１０匹には、表１９に示すように、実施例１で調製した１２種類の化合物のいずれかを有効成分として含有する製剤のいずれか１種を、３週齢から１０週齢まで、５６日間、１日１回、毎日、０．５ｍｌ／匹で腹腔内投与した（試験群１乃至１２）。残りの１群１０匹には、滅菌したマルトースの１０％水溶液（パイロジェンフリー）を、３週齢から１０週齢まで、５６日間、１日１回、毎日、０．５ｍｌ／匹で腹腔内投与した（対照群）。投与期間終了の翌日、実験２と同様に、各ハムスターの体重を測定し、ロタロッド試験、斜面耐久試験及び転倒回数の測定を行った。各群に投与した製剤の有効成分である化合物の種類と、測定結果とを表１９に示す。なお、対照群のハムスターの３週齢の平均体重は３５．４ｇ、１０週齢の平均体重は１２２．９ｇで、実施例１で調製した製剤を投与し試験験群１乃至１２の何れの群においても、その平均体重に対照群と有意の差は認められなかったので、表２５には、ロタロッド試験、斜面耐久試験及び転倒回数の測定結果のみを示す。 <Effects of cerebellar ataxia hamsters on motor coordination decline>
As in Experiment 2, 130 cerebellar ataxia hamsters born in the same week were randomly divided into 10 animals each in 13 groups. As shown in Table 19, in each of 10 groups of 12 groups, any one of the preparations containing any of the 12 compounds prepared in Example 1 as an active ingredient was administered from 3 weeks of age to 10 weeks. Until the age, it was intraperitoneally administered at 0.5 ml / mouse once a day for 56 days (test groups 1 to 12). The remaining 10 animals in a group were intraperitoneally administered with a 10% aqueous solution of sterile maltose (pyrogen-free) from 3 weeks to 10 weeks of age, once daily for 56 days, daily at 0.5 ml / mouse. (Control group). The day after the end of the administration period, the body weight of each hamster was measured in the same manner as in Experiment 2, and the rotarod test, the slope endurance test, and the number of falls were measured. Table 19 shows the types of compounds that are active ingredients of the preparations administered to each group and the measurement results. In addition, the average body weight of a 3-week-old hamster in the control group was 35.4 g, and the average body weight of 10-week-old was 122.9 g. No significant difference was found in the average body weight from the control group, and Table 25 shows only the results of the rotarod test, the slope endurance test, and the number of falls.
＜アミロイドβフラグメント投与マウス（アルツハイマー病モデル）に対する効果＞
　ＩＣＲマウス（日本チャールスリバー社販売）１３０匹を無作為に１０匹ずつ、１３群に分けた。実験３で使用した配列表における配列番号１で表されるアミノ酸配列を有するアミロイドβフラグメントを、３７℃で４日間エイジングさせて、１３０匹のマウスの側脳室内に９ｎｍｏｌ／６μｌ／匹投与した（投与法は、『Ｂｒａｉｎ　Ｒｅｓｅａｒｃｈ』、　第７０６巻、１８１－１９３頁（１９９６年）参照）。アミロイドβフラグメント投与後１日目から表２０に示すように、１２群各１０匹（試験群１３乃至２４）には、実施例１で調製した１２種類の化合物のいずれかを有効成分として含有する製剤のいずれか１種を、１日１回８日目まで毎日、０．３ｍｌ／匹、腹腔内投与した。残りの１群１０匹には、滅菌したマルトースの１０％水溶液（パイロジェンフリー）を１日１回８日目まで毎日、０．３ｍｌ／匹、腹腔内投与した（対照群）。アミロイドβフラグメント投与８日目に新奇物体認識試験（例えば、特開２００８－１９３９４１号公報参照）を行い、認知機能の指標として、各試験群における識別指数（全体の物体探索時間に対する新奇物体への探索時間延長の割合）の平均を求めて表２６に併せて示す。また、投与９日目にマウスを解剖して脳を採取し、常法により組織標本を作製して、アミロイドβフラグメント凝集物の沈着をコンゴーレッド染色もしくはチオフラビンＴ染色により確認すると同時に、ヘマトキシリン―エオジン染色もしくはニッスル染色した標本にて、認知機能にかかわる海馬領域の錐体細胞の変性もしくは脱落の度合いを観察した。海馬錐体細胞の変性もしくは脱落は、アミロイドβフラグメント非投与対照の状態を無（０）として、軽度（１）、中程度（２）、重度（３）の４段階で評価してスコア化し、各試験群１０匹のマウスの平均を求めて表２０に併せて示す。 <Effects on mice treated with amyloid β fragment (Alzheimer's disease model)>
130 ICR mice (available from Charles River Japan Co., Ltd.) were randomly divided into 13 groups, 10 each. The amyloid β fragment having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing used in Experiment 3 was aged at 37 ° C. for 4 days and administered into the lateral ventricle of 130 mice (9 nmol / 6 μl / mouse) ( The administration method is described in “Brain Research”, 706, 181-193 (1996)). As shown in Table 20 from the first day after administration of amyloid β fragment, each of 10 animals in 12 groups (test groups 13 to 24) contains any of the 12 compounds prepared in Example 1 as active ingredients. Any one of the formulations was intraperitoneally administered once a day until the 8th day, daily at 0.3 ml / animal. The remaining 10 animals in 1 group were intraperitoneally administered with a 10% aqueous solution of maltose (pyrogen-free) once a day until the 8th day (control group). A novel object recognition test (see, for example, Japanese Patent Application Laid-Open No. 2008-193941) is performed on the 8th day after administration of amyloid β fragment. The average of the search time extension ratio) is obtained and shown in Table 26 together. On day 9 of administration, mice were dissected and brains were collected, tissue specimens were prepared by a conventional method, and deposition of amyloid β fragment aggregates was confirmed by Congo red staining or thioflavin T staining, and at the same time, hematoxylin-eosin The degree of degeneration or occlusion of pyramidal cells in the hippocampal region involved in cognitive function was observed in the stained or Nissl stained specimens. Hippocampal pyramidal cell degeneration or loss is scored by assessing it in four stages: mild (1), moderate (2), and severe (3), with the amyloid β fragment non-administered control state being no (0), Table 20 shows the average of 10 mice in each test group.
＜新奇物体認識試験の方法＞
　実験装置（縦３０ｃｍ、横４５ｃｍ、高さ３０ｃｍのガラス箱）及びマウスが記憶する２つの対象物（ｏｂｊｅｃｔ）を用意した。試験前日、マウスを対象物のない状態で実験装置内を１０分間自由探索させて環境に馴化させておいた。試験当日、試行間隔６０分間で以下の２回の試行を行なった。１回目の試行では、２つの同一の対象物を実験装置の両端に置き、マウスを１０分間自由探索させた。２回目の試行では、１回目の試行で用いた対象物の１つを別種の対象物と置き換え、マウスを５分間自由探索させた。各試行において、対象物から１ｃｍ以内に鼻を近づけたり、対象物を鼻や髭で触れている状態を探索行動と定義して、その探索時間を計測した。物体識別指数（＝（新しい対象物の探索に費やした時間－一度記憶した対象物の探索に費やした時間）／（新しい対象物の探索に費やした時間＋一度記憶した対象物の探索に費やした時間））を求めた。この場合、識別指数は、新規対象物の探索により多く割かれた時間の全探索時間に対する割合であり、一度探索した対象物を動物が記憶していれば値が大きくなり、記憶していなければ値が小さくなる。 <Method of novel object recognition test>
An experimental apparatus (a glass box having a length of 30 cm, a width of 45 cm, and a height of 30 cm) and two objects stored by the mouse were prepared. The day before the test, the mouse was allowed to acclimatize to the environment by freely searching the experimental apparatus for 10 minutes in the absence of an object. On the test day, the following two trials were performed with a trial interval of 60 minutes. In the first trial, two identical objects were placed at both ends of the experimental apparatus and allowed to freely explore the mouse for 10 minutes. In the second trial, one of the objects used in the first trial was replaced with another kind of object, and the mouse was allowed to freely search for 5 minutes. In each trial, a state in which the nose was brought within 1 cm from the object or the object was touched with a nose or a heel was defined as a search action, and the search time was measured. Object identification index (= (time spent searching for a new object−time spent searching for an object once stored) / (time spent searching for a new object + searching for an object stored once) Time)). In this case, the identification index is the ratio of the time allotted to the search for new objects to the total search time, and the value increases if the animal stores the object once searched, and does not store it The value becomes smaller.
Figure JPOXMLDOC01-appb-T000073
Figure JPOXMLDOC01-appb-T000073
Figure JPOXMLDOC01-appb-T000074
Figure JPOXMLDOC01-appb-T000074
　表２５から明らかなように、実施例１で調製した１２種類の製剤は、いずれも、１０週齢の小脳失調症ハムスターの、ロタロッドからの落下時間低下、斜面耐久傾斜角度の低下及び転倒回数の増加を、顕著に改善した。１２種類の製剤を投与した場合の効果の強さを比較すると、何れの試験においても、ジメチン系スチリル色素化合物（ＮＫ－５２３、ＮＫ－５５７、及び、ＮＫ－１５１６）を含有する製剤を投与した場合（試験群１０乃至１２）よりも、ペンタメチン系シアニン色素化合物（ＮＫ－４、ＮＫ－２６、ＮＫ－２８、ＮＫ－１４７、ＮＫ－１９、ＮＫ－５３、ＮＫ－１５０、ＮＫ－３９３、Ｋ－１００、Ｋ－５２８、Ｋ－５５７、及び、ＮＫ－１５１６）を投与した場合（実験群１乃至９）の方が、強い運動協調性の改善効果が認められた。ペンタメチン系シアニン色素化合物間で、効果の強さを改善すると、ＮＫ－４、ＮＫ－２６、ＮＫ－１５０及びＮＫ－３９３で特に強い運動協調性の改善効果が認められた。この結果は、製造した製剤は、いずれも、神経変性疾患の治療剤として有用であることを物語っている。また、これらの製剤は、５６日間投与しても、ハムスターの体重は、対照群と有意な差が認められなかったので、いずれも安全性は高いと判断した。また、表２６から明らかなように、実施例１で調製した１２種類の製剤は、いずれも、アミロイドβフラグメント投与マウスの海馬錐体細胞の変性を抑制すると同時に、認知機能の低下を抑制した。１２種類の製剤を投与した場合の効果の強さを比較すると、いずれの試験においても、ジメチン系スチリル色素化合物（ＮＫ－５２３、ＮＫ－５５７、及び、ＮＫ－１５１６）を含有する製剤を投与した場合（実験群２２乃至２４）よりも、ペンタメチン系シアニン色素化合物（ＮＫ－４、ＮＫ－２６、ＮＫ－２８、ＮＫ－１４７、ＮＫ－１９、ＮＫ－５３、ＮＫ－１５０、ＮＫ－３９３、Ｋ－１００、Ｋ－５２８、Ｋ－５５７、及び、ＮＫ－１５１６）を投与した場合（実験群１３乃至２１）の方が、アミロイドβフラグメント投与による海馬錐体細胞の変性や認知機能障害に対して強い改善効果が認められた。 As is clear from Table 25, all of the 12 types of preparations prepared in Example 1 were those of 10-week-old cerebellar ataxia hamsters that had decreased fall time from rotarod, decreased slope endurance slope angle, and number of falls. Increase was significantly improved. Comparing the strength of the effect when 12 types of preparations were administered, in any test, preparations containing dimethine styryl dye compounds (NK-523, NK-557, and NK-1516) were administered. More than the cases (test groups 10 to 12), pentamethine cyanine dye compounds (NK-4, NK-26, NK-28, NK-147, NK-19, NK-53, NK-150, NK-393, K When -100, K-528, K-557, and NK-1516) were administered (Experimental groups 1 to 9), a stronger effect of improving motor coordination was observed. When the strength of the effect was improved among the pentamethine cyanine dye compounds, NK-4, NK-26, NK-150 and NK-393 showed a particularly strong effect of improving motor coordination. This result indicates that any of the prepared preparations is useful as a therapeutic agent for neurodegenerative diseases. In addition, even when these preparations were administered for 56 days, the hamster body weight was not significantly different from that of the control group. Moreover, as is clear from Table 26, all of the 12 types of preparations prepared in Example 1 suppressed degeneration of hippocampal pyramidal cells of mice administered with amyloid β fragment and at the same time suppressed cognitive decline. Comparing the strength of the effect when 12 kinds of preparations were administered, in any test, preparations containing dimethine styryl dye compounds (NK-523, NK-557, and NK-1516) were administered. More than the case (experimental groups 22 to 24), pentamethine cyanine dye compounds (NK-4, NK-26, NK-28, NK-147, NK-19, NK-53, NK-150, NK-393, K -100, K-528, K-557, and NK-1516) (Experimental Groups 13 to 21) were more effective against hippocampal pyramidal cell degeneration and cognitive impairment due to amyloid β fragment administration A strong improvement effect was observed.
＜注射用の液剤＞
　注射用精製水３７０ｇに注射用精製マルトース（株式会社林原製造）６０ｇを溶解した溶液と、注射用精製水１７０ｇに、レシチン２ｇと有効成分として、ＮＫ－４（化学式２で表される化合物）、ＮＫ－２３４（一般式２で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が３である化合物）、ＮＫ－２６（化学式１で表される化合物）、ＮＫ－２８（一般式２で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が７である化合物）、ＮＫ－１４７（一般式２で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が８である化合物）、ＮＫ－１９（化学式４で表される化合物）、ＮＫ－５３（化学式５で表される化合物）、ＮＫ－１５０（化学式３で表される化合物）、ＮＫ－３９３（一般式３で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が８である化合物）、ＮＫ－１００（化学式６で表される化合物）ＮＫ－５２８（化学式７で表される化合物）、ＮＫ－５５７（化学式８で表される化合物）、及び、ＮＫ－１５１６（化学式９で表される化合物）（いずれも株式会社林原生物化学研究所製造）のいずれか１種を、各々１２０ｍｇ溶解した溶液とを混合し、濾過滅菌後、溶存する酸素の濃度が約０．１ｐｐｍになるまで無菌の窒素ガスをバブリングして、褐色アンプルに１ｍｌずつ分注し、窒素気流下でアンプルを封止した。本品は、いずれもパイロジェンフリーであり、抗神経変性疾患剤として利用できる。また、本品は、神経変性抑制剤、神経細胞保護剤、神経突起促進剤や、神経変性に伴う病態や神経機能障害の治療剤としても利用できる。また、本品は脳保護剤、脳の酸化的障害抑制剤、虚血性脳障害抑制剤、脳梗塞巣進展抑制剤、脳浮腫抑制剤、遅発性神経死抑制剤、脳機能正常化剤、酸化ストレス抑制剤、抗潰瘍剤、血糖上昇抑制剤、眼性疾患の予防・治療剤、移植臓器保存剤、移植組織・臓器の壊死防止剤、組織・臓器の障害の予防・治療剤、放射線障害予防・治療剤、抗腫瘍剤、腫瘍転移抑制剤、細胞障害マーカー抑制剤、炎症性疾患やそれに伴う組織障害の予防・治療剤、感覚細胞、感覚神経或いは感覚器の障害の抑制剤、薬物中毒の予防・治療剤、カルシウム・ナトリウム交換系阻害剤、疼痛や掻痒の予防・治療剤、プロテインキナーゼ刺激剤、ミトコンドリア脳筋症予防・治療剤、動脈閉塞・狭窄予防・治療剤、血液脳関門破綻抑制剤、薬物依存症治療剤、アポトーシス抑制剤、過酸化脂質生成抑制剤、ラジカルスカベンジャー、アミロイドβペプチド凝集阻害剤、アミロイドβペプチド傷害抑制剤、コリンエステラーゼ活性阻害剤、セリン／スレオニンキナーゼ（Ａｋｔ）活性化剤、ホスファチヂルイノシトール（３，４，５）３リン酸キナーゼ（ＰＩ３Ｋ）－セリン／スレオニンキナーゼ（Ａｋｔ）カスケード活性化剤、サイクリックＡＭＰ濃度上昇促進剤、或いは、ＳＡＰＫ／ＪＮＫリン酸化抑制剤として利用してもよい。さらに、本発明の抗神経変性疾患剤は、神経変性疾患を発症したペットをはじめとするヒト以外の動物の治療剤や、その予防剤として使用することもできる。 <Liquid preparation for injection>
A solution prepared by dissolving 60 g of purified maltose for injection (manufactured by Hayashibara Co., Ltd.) in 370 g of purified water for injection, and 2 g of lecithin and an active ingredient NK-4 (compound represented by Chemical Formula 2) in 170 g of purified water for injection, NK-234 (compound in which the alkyl group (R) in the side chain of the compound represented by general formula 2 has 3 carbon atoms), NK-26 (compound represented by chemical formula 1), NK-28 (general formula 2), NK-147 (the number of carbon atoms in the side chain alkyl group (R) of the compound represented by the general formula 2 is 7). 8), NK-19 (compound represented by chemical formula 4), NK-53 (compound represented by chemical formula 5), NK-150 (compound represented by chemical formula 3), NK-393 (general The side chain alkyl group of the compound represented by Formula 3 ( )), NK-100 (compound represented by chemical formula 6), NK-528 (compound represented by chemical formula 7), NK-557 (compound represented by chemical formula 8), and , NK-1516 (compound represented by the chemical formula 9) (all manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) were mixed with a solution in which 120 mg each was dissolved, and after filtration sterilization, dissolved oxygen Aseptic nitrogen gas was bubbled until the concentration reached about 0.1 ppm, and 1 ml each was dispensed into a brown ampule, and the ampule was sealed under a nitrogen stream. All of this product is pyrogen-free and can be used as an anti-neurodegenerative disease agent. In addition, the product can be used as a neurodegeneration inhibitor, a nerve cell protective agent, a neurite promoter, or a therapeutic agent for a disease state or neurological dysfunction associated with neurodegeneration. In addition, this product is a brain protective agent, brain oxidative disorder inhibitor, ischemic brain disorder inhibitor, cerebral infarction growth inhibitor, brain edema inhibitor, delayed neuronal death inhibitor, brain function normalizing agent, Oxidative stress inhibitor, anti-ulcer agent, blood sugar elevation inhibitor, prevention / treatment agent for ocular diseases, transplanted organ preservative, transplanted tissue / organ necrosis inhibitor, tissue / organ injury preventive / therapeutic agent, radiation damage Prophylactic / therapeutic agent, antitumor agent, tumor metastasis inhibitor, cytopathic marker inhibitor, prophylactic / therapeutic agent for inflammatory diseases and associated tissue disorders, sensory cell, sensory nerve or sensory organ disorder inhibitor, drug addiction Preventive / therapeutic agent, calcium / sodium exchange inhibitor, preventive / therapeutic agent for pain and pruritus, protein kinase stimulator, preventive / therapeutic agent for mitochondrial encephalomyopathy, preventive / therapeutic agent for arterial occlusion / stenosis, blood-brain barrier disruption Inhibitors, drug dependence treatments, Apot Cis inhibitor, lipid peroxide production inhibitor, radical scavenger, amyloid β peptide aggregation inhibitor, amyloid β peptide injury inhibitor, cholinesterase activity inhibitor, serine / threonine kinase (Akt) activator, phosphatidylinositol (3 , 4, 5) 3-phosphate kinase (PI3K) -serine / threonine kinase (Akt) cascade activator, cyclic AMP concentration increase promoter, or SAPK / JNK phosphorylation inhibitor. Furthermore, the anti-neurodegenerative disease agent of the present invention can also be used as a therapeutic agent for a non-human animal including a pet that has developed a neurodegenerative disease, or as a preventive agent thereof.
　実施例２で調製した１３種類の抗神経変性疾患剤を、各々、１０匹のｄｄｙマウス（平均体重２５．６ｇ）の腹腔内に、０．５ｍｌ／匹単回投与して、投与後１週間、毎日体重を測定しながら、経過を観察したところ、対照として１０匹のｄｄｙマウス（平均体重２６．３ｇ）に、０．２％のレシチンを含む１０％マルトース溶液を腹区内に投与して、経過を観察した場合に比して、体重に有意な変化は認められず、他に外観的な変化も認められなかった。この結果は、実施例２で調製した１３種類の抗神経変性疾患剤の有効成分として配合した化合物のＬＤ５０は、いずれも３．９ｍｇ／ｋｇ・体重以上となるので、これらの製剤がヒトに投与しても安全な製剤であることを物語っている。 Each of the 13 types of anti-neurodegenerative disease agents prepared in Example 2 was administered as a single 0.5 ml / mouse intraperitoneally to 10 ddy mice (average body weight 25.6 g) for 1 week after administration. As a control, 10 dmal mice (average body weight 26.3 g) were administered with 10% maltose solution containing 0.2% lecithin intraperitoneally as a control. Compared with the observation of the course, no significant change in body weight was observed, and no other change in appearance was observed. As a result, the LD 50 of the compound formulated as an active ingredient of the 13 types of anti-neurodegenerative disease agents prepared in Example 2 is 3.9 mg / kg · body weight or more. It shows that it is safe to administer.
＜注射用の粉末剤＞
　注射用精製水３７０ｇに注射用精製マルトース（株式会社林原製造）６０ｇを溶解した溶液と、注射用精製水１７０ｇに、ポリソルベイト８０（日本油脂株式会社販売）３ｇと、有効成分として、ＮＫ－４（化学式２で表される化合物）、ＮＫ－２３４（一般式２で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が３である化合物）、ＮＫ－２６（化学式１で表される化合物）、ＮＫ－２８（一般式２で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が７である化合物）、ＮＫ－１４７（一般式２で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が８である化合物）、ＮＫ－１９（化学式４で表される化合物）、ＮＫ－５３（化学式５で表される化合物）、ＮＫ－１５０（化学式３で表される化合物）、ＮＫ－３９３（一般式３で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が８である化合物）、ＮＫ－１００（化学式６で表される化合物）ＮＫ－５２８（化学式７で表される化合物）、ＮＫ－５５７（化学式８で表される化合物）、及び、ＮＫ－１５１６（化学式９で表される化合物）（いずれも株式会社林原生物化学研究所製造）のいずれか１種を、各々６０ｍｇ溶解した溶液とを混合して濾過滅菌後、褐色アンプルに１０ｍｌずつ分注し、常法により凍結乾燥後、窒素気流下でアンプルを封止した。本品は、いずれもパイロジェンフリーであり、用時に、アンプルに注射用精製水乃至生理食塩水２乃至１０ｍｌを加えて溶解し、点滴静注、皮下投与、腹腔内投与などの方法で使用する。本品は、抗神経変性疾患剤として利用できる。また、本品は、神経変性抑制剤、神経細胞保護剤、神経突起促進剤や、神経変性に伴う病態や神経機能障害の治療剤としても利用できる。また、本品は脳保護剤、脳の酸化的障害抑制剤、虚血性脳障害抑制剤、脳梗塞巣進展抑制剤、脳浮腫抑制剤、遅発性神経死抑制剤、脳機能正常化剤、酸化ストレス抑制剤、抗潰瘍剤、血糖上昇抑制剤、眼性疾患の予防・治療剤、移植臓器保存剤、移植組織・臓器の壊死防止剤、組織・臓器の障害の予防・治療剤、放射線障害予防・治療剤、抗腫瘍剤、腫瘍転移抑制剤、細胞障害マーカー抑制剤、炎症性疾患やそれに伴う組織障害の予防・治療剤、感覚細胞、感覚神経或いは感覚器の障害の抑制剤、薬物中毒の予防・治療剤、カルシウム・ナトリウム交換系阻害剤、疼痛や掻痒の予防・治療剤、プロテインキナーゼ刺激剤、ミトコンドリア脳筋症予防・治療剤、動脈閉塞・狭窄予防・治療剤、血液脳関門破綻抑制剤、薬物依存症治療剤、アポトーシス抑制剤、過酸化脂質生成抑制剤、ラジカルスカベンジャー、アミロイドβペプチド凝集阻害剤、アミロイドβペプチド傷害抑制剤、コリンエステラーゼ活性阻害剤、セリン／スレオニンキナーゼ（Ａｋｔ）活性化剤、ホスファチヂルイノシトール（３，４，５）３リン酸キナーゼ（ＰＩ３Ｋ）－セリン／スレオニンキナーゼ（Ａｋｔ）カスケード活性化剤、サイクリックＡＭＰ濃度上昇促進剤、或いは、ＳＡＰＫ／ＪＮＫリン酸化抑制剤として利用してもよい。さらに、本発明の抗神経変性疾患剤は、神経変性疾患を発症したペットをはじめとするヒト以外の動物の治療剤や、その予防剤として使用することもできる。 <Powder for injection>
A solution prepared by dissolving 60 g of purified maltose for injection (manufactured by Hayashibara Co., Ltd.) in 370 g of purified water for injection, 170 g of purified water for injection, 3 g of polysorbate 80 (sold by NOF Corporation), Compound represented by chemical formula 2), NK-234 (compound in which the carbon number of the alkyl group (R) in the side chain of the compound represented by general formula 2 is 3), NK-26 (represented by chemical formula 1) Compound), NK-28 (a compound in which the alkyl group (R) in the side chain of the compound represented by Formula 2 has 7 carbon atoms), NK-147 (a compound in the side chain of the compound represented by Formula 2) A compound in which the alkyl group (R) has 8 carbon atoms), NK-19 (a compound represented by Formula 4), NK-53 (a compound represented by Formula 5), NK-150 (a compound represented by Formula 3) Compound), NK-393 (general formula 3) A compound in which the carbon number of the alkyl group (R) in the side chain of the compound is 8), NK-100 (a compound represented by Chemical Formula 6), NK-528 (a compound represented by Chemical Formula 7), NK— A solution in which 60 mg of each of 557 (compound represented by chemical formula 8) and NK-1516 (compound represented by chemical formula 9) (both manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) were dissolved, After sterilizing by filtration, 10 ml each was dispensed into brown ampules, freeze-dried by a conventional method, and sealed in a nitrogen stream. All of these products are pyrogen-free. At the time of use, 2 to 10 ml of purified water for injection or physiological saline is added to the ampoule and dissolved, and then used by methods such as intravenous infusion, subcutaneous administration, and intraperitoneal administration. This product can be used as an anti-neurodegenerative disease agent. In addition, the product can be used as a neurodegeneration inhibitor, a nerve cell protective agent, a neurite promoter, or a therapeutic agent for a disease state or neurological dysfunction associated with neurodegeneration. In addition, this product is a brain protective agent, brain oxidative disorder inhibitor, ischemic brain disorder inhibitor, cerebral infarction growth inhibitor, brain edema inhibitor, delayed neuronal death inhibitor, brain function normalizing agent, Oxidative stress inhibitor, anti-ulcer agent, blood sugar elevation inhibitor, prevention / treatment agent for ocular diseases, transplanted organ preservative, transplanted tissue / organ necrosis inhibitor, tissue / organ injury preventive / therapeutic agent, radiation damage Prophylactic / therapeutic agent, antitumor agent, tumor metastasis inhibitor, cytopathic marker inhibitor, prophylactic / therapeutic agent for inflammatory diseases and associated tissue disorders, sensory cell, sensory nerve or sensory organ disorder inhibitor, drug addiction Preventive / therapeutic agent, calcium / sodium exchange inhibitor, preventive / therapeutic agent for pain and pruritus, protein kinase stimulator, preventive / therapeutic agent for mitochondrial encephalomyopathy, preventive / therapeutic agent for arterial occlusion / stenosis, blood-brain barrier disruption Inhibitors, drug dependence treatments, Apot Cis inhibitor, lipid peroxide production inhibitor, radical scavenger, amyloid β peptide aggregation inhibitor, amyloid β peptide injury inhibitor, cholinesterase activity inhibitor, serine / threonine kinase (Akt) activator, phosphatidylinositol (3 , 4, 5) 3-phosphate kinase (PI3K) -serine / threonine kinase (Akt) cascade activator, cyclic AMP concentration increase promoter, or SAPK / JNK phosphorylation inhibitor. Furthermore, the anti-neurodegenerative disease agent of the present invention can also be used as a therapeutic agent for a non-human animal including a pet that has developed a neurodegenerative disease, or as a preventive agent thereof.
　本発明の抗神経変性疾患剤は、神経細胞の神経変性に起因するパーキンソン病、パーキンソン症候群、アルツハイマー病、認知症、又は、脳卒中などの予防、治療及び／又は進展抑制、さらには、神経変性を引き起こす因子からの神経細胞保護に有用である。また、神経変性疾患に伴う種々の病態や神経機能障害（例えば、振戦、固縮、無動、寡動、動作緩慢、姿勢反射障害、自律神経障害、突進現象、歩行障害、うつ、記憶障害、筋萎縮、筋力低下、上肢機能障害、構音障害、嚥下障害、呼吸障害、しびれ及び麻痺など）の改善にも有用である。しかも、本発明の抗神経変性疾患剤は、長期間投与しても、副作用がないので、安全性が高く、安心して利用することができる。本発明は、斯くも顕著な作用効果を奏する発明であり、斯界に多大の貢献をする、誠に意義のある発明である。 The anti-neurodegenerative disease agent of the present invention prevents, treats and / or suppresses progression of Parkinson's disease, Parkinson's syndrome, Alzheimer's disease, dementia, or stroke caused by neurodegeneration of nerve cells, and further suppresses neurodegeneration. Useful for protecting nerve cells from causing factors. In addition, various pathological conditions and neurological dysfunctions associated with neurodegenerative diseases (for example, tremor, rigidity, ataxia, peristalsis, slow movement, posture reflex disorder, autonomic disorder, lunging phenomenon, gait disorder, depression, memory disorder, It is also useful for improving muscle atrophy, muscle weakness, upper limb dysfunction, articulation disorder, dysphagia, respiratory disorder, numbness and paralysis. Moreover, since the anti-neurodegenerative disease agent of the present invention has no side effects even when administered for a long period of time, it is highly safe and can be used with confidence. The present invention is an invention that exhibits such remarkable effects, and is a truly significant invention that contributes greatly to the world.

Claims (10)

  1. 　一般式１で表される化合物を有効成分として含有する抗神経変性疾患剤。
    Figure JPOXMLDOC01-appb-C000001
     （一般式１におけるＲ１乃至Ｒ３は、それぞれ独立に、水素原子又は適宜の置換基を表し、Ｚ１は複素環を、また、Ｚ２はＺ１と同じか異なる複素環又は芳香環を表し、それらの複素環及び芳香環は置換基を有していてもよい。ｏは０又は１、２のいずれかである整数を表し、ｐは、０又は１のいずれかである整数を表し、ｏが０又は２のとき、ｐは１であり、ｏが１のとき、ｐは０である。ｏが０の場合、Ｒ１、Ｒ２は存在せず、ｐが０の場合、Ｒ３は存在せず、Ｒ２が結合する炭素とＺ２とは一重結合となる。Ｘｌ －は適宜の対アニオンを表し、ｑは１又は２のいずれかである整数を表す。）     An anti-neurodegenerative disease agent comprising a compound represented by the general formula 1 as an active ingredient.
    Figure JPOXMLDOC01-appb-C000001
     (R 1 to R 3 in General Formula 1 each independently represents a hydrogen atom or an appropriate substituent, Z 1 represents a heterocyclic ring, and Z 2 represents a heterocyclic ring or an aromatic ring that is the same as or different from Z 1. And the heterocyclic ring and the aromatic ring may have a substituent, o represents an integer of 0, 1 or 2, and p represents an integer of 0 or 1. , O is 0 or 2, p is 1, and when o is 1, p is 0. When o is 0, R 1 and R 2 do not exist, and when p is 0, R 3 is not present, the single bond between carbon and Z 2 wherein R 2 is attached .X l - represents a suitable counter anion, q is an integer of either 1 or 2).
  2. 　一般式１で表される化合物が、一般式２乃至５のいずれかで表される化合物である請求の範囲第１項記載の抗神経変性疾患剤。
    Figure JPOXMLDOC01-appb-C000002
     （一般式２において、Ｒ４乃至Ｒ６は互いに同じか異なる脂肪族炭化水素基を表す。Ｘ２ －は適宜の対アニオンを表し、ｍはカチオン部の電荷とバランスする電荷となる１又は２のいずれかである整数を表す。）
    Figure JPOXMLDOC01-appb-C000003
     （一般式３において、Ｒ７乃至Ｒ９は互いに同じか異なる脂肪族炭化水素基を表す。Ｘ３ －は適宜の対アニオンを表し、ｍはカチオン部の電荷とバランスする電荷となる１又は２のいずれかである整数を表す。）
    Figure JPOXMLDOC01-appb-C000004
     （一般式４において、Ｒ１０乃至Ｒ１２は互いに同じか異なる脂肪族炭化水素基を表す。Ｘ４ －は適宜の対アニオンを表し、ｍはカチオン部の電荷とバランスする電荷となる１又は２のいずれかである整数を表す。）
    Figure JPOXMLDOC01-appb-C000005
     （一般式５において、Ｚ３は複素芳香環を表し、その複素芳香環は置換基を有していてもよい。Ｚ４は芳香環又は複素芳香環を表し、それらの複素芳香環及び芳香環は置換基を有していてもよい。Ｒ１３は脂肪族炭化水素基を表し、その脂肪族炭化水素基は置換基を有していてもよい。Ｒ１４は水素原子又は適宜の置換基を、また、Ｘ５ －は適宜の対アニオンを表す。）     The anti-neurodegenerative disease agent according to claim 1, wherein the compound represented by the general formula 1 is a compound represented by any one of the general formulas 2 to 5.
    Figure JPOXMLDOC01-appb-C000002
     (In the general formula 2, R 4 to R 6 are the same or different aliphatic hydrocarbon radicals each other .X 2 - represents an appropriate counter anion, m is 1 or 2 as a charge balancing the charge of the cation portion Represents an integer that is either
    Figure JPOXMLDOC01-appb-C000003
     (In General Formula 3, R 7 to R 9 represent the same or different aliphatic hydrocarbon groups. X 3 — represents an appropriate counter anion, and m represents a charge balanced with the charge of the cation moiety. Represents an integer that is either
    Figure JPOXMLDOC01-appb-C000004
     (In general formula 4, R 10 to R 12 are the same or different aliphatic hydrocarbon radicals each other .X 4 - represents a suitable counter anion, m is 1 or 2 as a charge balancing the charge of the cation portion Represents an integer that is either
    Figure JPOXMLDOC01-appb-C000005
     (In General Formula 5, Z 3 represents a heteroaromatic ring, and the heteroaromatic ring may have a substituent. Z 4 represents an aromatic ring or a heteroaromatic ring, and these heteroaromatic ring and aromatic ring R 13 represents an aliphatic hydrocarbon group, the aliphatic hydrocarbon group may have a substituent, and R 14 represents a hydrogen atom or an appropriate substituent. in addition, X 5 - represents a suitable counter anion).
  3. 　一般式１乃至５のいずれかで表される化合物の対アニオンが、沃素イオン又は塩素イオンである請求の範囲第１項又は第２項記載の抗神経変性疾患剤。 3. The anti-neurodegenerative disease agent according to claim 1 or 2, wherein the counter anion of the compound represented by any one of the general formulas 1 to 5 is an iodine ion or a chlorine ion.
  4. 　一般式２で表される化合物が化学式２で表される化合物であり、一般式３で表される化合物が化学式３で表される化合物である請求の範囲第２項記載の抗神経変性疾患剤。
    Figure JPOXMLDOC01-appb-C000006
    Figure JPOXMLDOC01-appb-C000007
         The anti-neurodegenerative disease agent according to claim 2, wherein the compound represented by the general formula 2 is a compound represented by the chemical formula 2, and the compound represented by the general formula 3 is a compound represented by the chemical formula 3. .
    Figure JPOXMLDOC01-appb-C000006
    Figure JPOXMLDOC01-appb-C000007
  5. 　製剤学的に許容される１種又は２種以上の成分を含んでなる請求の範囲第１項乃至第４項のいずれかに記載の抗神経変性疾患剤。 The anti-neurodegenerative disease agent according to any one of claims 1 to 4, comprising one or more pharmaceutically acceptable components.
  6. 　製剤学的に許容される成分が、水性媒体である請求の範囲第５項記載の抗神経変性疾患剤。 The anti-neurodegenerative disease agent according to claim 5, wherein the pharmaceutically acceptable ingredient is an aqueous medium.
  7. 　抗神経変性疾患剤が、神経細胞変性抑制剤、神経細胞保護剤、又は、神経細胞変性に伴う運動失調症改善剤である請求の範囲第１項乃至第６項のいずれかに記載の抗神経変性疾患剤。 The anti-nerve according to any one of claims 1 to 6, wherein the anti-neurodegenerative disease agent is a nerve cell degeneration inhibitor, a nerve cell protective agent, or an ataxia ameliorating agent associated with nerve cell degeneration. Degenerative disease agent.
  8. 　神経が小脳プルキンエ細胞である請求の範囲第１項乃至第７項のいずれかに記載の抗神経変性疾患剤。 The anti-neurodegenerative disease agent according to any one of claims 1 to 7, wherein the nerve is a cerebellar Purkinje cell.
  9. 　神経変性疾患が、パーキンソン病、認知症、脊髄小脳変性症、アルツハイマー病、脳梗塞、又は、運動失調症である請求の範囲第１項乃至第６項のいずれかに記載の抗神経変性疾患剤。 The anti-neurodegenerative disease agent according to any one of claims 1 to 6, wherein the neurodegenerative disease is Parkinson's disease, dementia, spinocerebellar degeneration, Alzheimer's disease, cerebral infarction, or ataxia. .
  10. 　脳保護剤、脳の酸化的障害抑制剤、虚血性脳障害抑制剤、脳梗塞巣進展抑制剤、脳浮腫抑制剤、遅発性神経死抑制剤、脳機能正常化剤、酸化ストレス抑制剤、抗潰瘍剤、血糖上昇抑制剤、眼性疾患の予防・治療剤、移植臓器保存剤、移植組織・臓器の壊死防止剤、組織・臓器の障害の予防・治療剤、放射線障害予防・治療剤、抗腫瘍剤、腫瘍転移抑制剤、細胞障害マーカー抑制剤、炎症性疾患やそれに伴う組織障害の予防・治療剤、感覚器の障害の抑制剤、薬物中毒の予防・治療剤、カルシウム・ナトリウム交換系阻害剤、疼痛や掻痒の予防・治療剤、プロテインキナーゼ刺激剤、ミトコンドリア脳筋症予防・治療剤、動脈閉塞・狭窄予防・治療剤、血液脳関門破綻抑制剤、薬物依存症治療剤、アポトーシス抑制剤、過酸化脂質生成抑制剤、ラジカルスカベンジャー、アミロイドβペプチド凝集阻害剤、アミロイドβペプチド傷害抑制剤、アセチルコリンエステラーゼ活性阻害剤、セリン／スレオニンキナーゼ（Ａｋｔ）活性化剤、ホスファチヂルイノシトール（３，４，５）３リン酸キナーゼ（ＰＩ３Ｋ）－セリン／スレオニンキナーゼ（Ａｋｔ）カスケード活性化剤、サイクリックＡＭＰ濃度上昇促進剤又は、ＳＡＰＫ／ＪＮＫリン酸化抑制剤としての請求の範囲第１項乃至第９項のいずれかに記載の抗神経変性疾患剤。 Brain protective agent, brain oxidative disorder inhibitor, ischemic brain disorder inhibitor, cerebral infarction growth inhibitor, brain edema inhibitor, delayed neuronal death inhibitor, brain function normalizer, oxidative stress inhibitor, Anti-ulcer agent, antihyperglycemic agent, preventive / therapeutic agent for ophthalmic diseases, transplanted organ preservative, transplanted tissue / organ necrosis inhibitor, preventive / therapeutic agent for tissue / organ injury, preventive / therapeutic agent for radiation injury, Antitumor agent, tumor metastasis inhibitor, cytopathic marker inhibitor, prophylactic / therapeutic agent for inflammatory diseases and associated tissue disorders, sensory organ disorder inhibitor, drug addiction preventive / therapeutic agent, calcium / sodium exchange system Inhibitors, pain and pruritus prevention / treatment agents, protein kinase stimulants, mitochondrial encephalomyopathy prevention / treatment agents, arterial occlusion / stenosis prevention / treatment agents, blood brain barrier breakdown inhibitors, drug dependence treatment agents, apoptosis inhibitors Agent, lipid peroxide production inhibitor Radical scavenger, amyloid β peptide aggregation inhibitor, amyloid β peptide injury inhibitor, acetylcholinesterase activity inhibitor, serine / threonine kinase (Akt) activator, phosphatidylinositol (3,4,5) triphosphate kinase ( The antitumor according to any one of claims 1 to 9, which is a PI3K) -serine / threonine kinase (Akt) cascade activator, a cyclic AMP concentration increase promoter, or a SAPK / JNK phosphorylation inhibitor. Neurodegenerative disease agent.
PCT/JP2010/050903 2009-01-29 2010-01-25 Anti-neurodegenerative disease agent WO2010087306A1 (en)

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